Modified vitamin K-dependent polypeptides

ABSTRACT

The invention provides vitamin k-dependent polypeptides with enhanced membrane binding affinity. These polypeptides can be used to modulate clot formation in mammals. Methods of modulating clot formation in mammals are also described.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 09/302,239, filedApr. 29, 1999, which is a continuation-in-part of U.S. Ser. No.08/955,636, filed on Oct. 23, 1997, now issued as U.S. Pat. No.6,017,882.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

Funding for work described herein was provided by the federalgovernment, which has certain rights in the invention.

BACKGROUND OF THE INVENTION

Vitamin K-dependent proteins contain 9 to 13 gamma-carboxyglutamic acidresidues (Gla) in their amino terminal 45 residues. The Gla residues areproduced by enzymes in the liver that utilize vitamin K to carboxylatethe side chains of glutamic acid residues in protein precursors. VitaminK-dependent proteins are involved in a number of biological processes,of which the most well-described is blood coagulation (reviewed inFurie, B. and Furie, B. C., 1988, Cell, 53:505-518). Vitamin K-dependentproteins include protein Z, protein S, prothrombin, factor X, factor IX,protein C, factor VII and Gas6. The latter protein functions in cellgrowth regulation. Matsubara et al., 1996, Dev. Biol., 180:499-510. TheGla residues are needed for proper calcium binding and membraneinteraction by these proteins. The membrane contact site of factor X isthought to reside within amino acid residues 1-37. Evans and Nelsestuen,1996, Protein Science 5:suppl. 1, 163 Abs. Although the Gla-containingregions of the plasma proteins show a high degree of sequence homology,they have at least a 1000-fold range in membrane affinity. McDonald, J.F. et al., 1997, Biochemistry, 36:5120-5137.

Factor VII functions in the initial stage of blood clotting and may be akey element in forming blood clots. The inactive precursor, or zymogen,has low enzyme activity that is greatly increased by proteolyticcleavage to form factor VIIa. This activation can be catalyzed by factorXa as well as by VIIa-tissue factor, an integral membrane protein foundin a number of cell types. Fiore, M. M., et al., 1994, J. Biol. Chem.,269:143-149. Activation by VIIa-tissue factor is referred to asautoactivation. It is implicated in both the activation (formation offactor VIIa from factor VII) and the subsequent activity of factor VIIa.The most important pathway for activation in vivo is not known. FactorVIIa can activate blood clotting factors IX and X.

Tissue factor is expressed at high levels on the surface of some tumorcells. A role for tissue factor, and for factor VIIa, in tumordevelopment and invasion of tissues is possible. Vrana, J. A. et al.,Cancer Res., 56:5063-5070. Cell expression and action of tissue factoris also a major factor in toxic response to endotoxic shock. Dackiw, A.A. et al., 1996, Arch. Surg., 131:1273-1278.

Protein C is activated by thrombin in the presence of thrombomodulin, anintegral membrane protein of endothelial cells. Esmon, N. L. et al.,1982, J. Biol. Chem., 257:859-864. Activated protein C (APC) degradesfactors Va and VIIIa in combination with its cofactor, protein S.Resistance to APC is the most common form of inherited thrombosisdisease. Dahlback, B., 1995, Blood, 85:607-614. Vitamin k inhibitors arecommonly administered as a prophylaxis for thrombosis disease.

Vitamin k-dependent proteins are used to treat certain types ofhemophilia. Hemophilia A is characterized by the absence of activefactor VIII, factor VIIIa, or the presence of inhibitors to factor VIII.Hemophilia B is characterized by the absence of active factor IX, factorIXa. Factor VII deficiency, although rare, responds well to factor VIIadministration. Bauer, K. A., 1996, Haemostasis, 26:155-158, suppl. 1.Factor VIII replacement therapy is limited due to development ofhigh-titer inhibitory factor VIII antibodies in some patients.Alternatively, factor VIIa can be used in the treatment of hemophilia Aand B. Factor IXa and factor VIIIa activate factor X. Factor VIIaeliminates the need for factors IX and VIII by activating factor Xdirectly, and can overcome the problems of factor IX and VIIIdeficiencies with few immunological consequences. Hedner et al., 1993,Transfus. Medi. Rev., 7:78-83; Nicolaisen, E. M. et al., 1996, Thromb.Haemost., 76:200-204. Effective levels of factor VIIa administration areoften high (45 to 90 μg/kg of body weight) and administration may needto be repeated every few hours. Shulmav, S. et al., 1996, Thromb.Haemost., 75:432-436.

A soluble form of tissue factor (soluble tissue factor or sTF) that doesnot contain the membrane contact region, has been found to beefficacious in treatment of hemophilia when co-administered with factorVIIa. U.S. Pat. No. 5,504,064. In dogs, sTF was shown to reduce theamount of factor VIIa needed to treat hemophilia. Membrane associationby sTF-VIIa is entirely dependent on the membrane contact site of factorVII. This contrasts to normal tissue-factor VIIa complex, which is boundto the membrane through both tissue factor and VII(a).

SUMMARY OF THE INVENTION

It has been discovered that modifications within the γ-carboxyglutamicacid (GLA) domain of vitamin K-dependent polypeptides enhance theirmembrane binding affinities. Vitamin K-dependent polypeptides modifiedin such a manner have enhanced activity and may be used asanti-coagulants, pro-coagulants or for other functions that utilizevitamin k-dependent proteins. For example, an improved factor VIImolecule may provide several benefits by lowering the dosage of VIIaneeded, the relative frequency of administration and/or by providingqualitative changes that allow more effective treatment of deficiencystates.

The invention features vitamin k-dependent polypeptides that include amodified GLA domain that enhances membrane binding affinity of thepolypeptide relative to a corresponding native vitamin k-dependentpolypeptide. The modified GLA domain is from about amino acid 1 to aboutamino acid 45 and includes at least one amino acid substitution. Forexample, the amino acid substitution can be at amino acid 11, 12, 29, 33or 34. Preferably, the substitution is at amino acid 11, 33, or 34. Themodified GLA domain may include an amino acid sequence which, in thecalcium saturated state, forms a tertiary structure having a cationiccore with a halo of electronegative charge.

The vitamin k-dependent polypeptide may be, for example, protein C,activated protein C, factor IX, factor IXa, factor VII, factor VIIa oractive site modified factor VIIa. The modified GLA domain of protein Cor activated protein C may include a glutamic acid residue at amino acid33 and an aspartic acid residue at amino acid 34. The modified GLAdomain of protein C or activated protein C may also include a glutamineor glutamic acid residue at amino acid 11. Additionally, a glycineresidue may be substituted at amino acid 12 in the GLA domain of proteinC or activated protein C.

The modified GLA domain of factor VII, factor VIIa, active site modifiedfactor VIIa, factor IX, and factor IXa may contain a substitution atamino acid 11, 29, 33, or combinations thereof. For example, themodified GLA domain may contain substitutions at residues 11 and 29, 11and 33, 29 and 33, or 11, 29, and 33. The modified GLA domain cancontain, for example, a substitution of a glutamine, a glutamic acid, anaspartic acid, or an asparagine residue at residue 11, and further caninclude a substitution at residue 29 such as substitution of a glutamicacid or a phenylalanine residue or an amino acid substitution at residue33 such as a glutamic acid or an aspartic acid residue. The modified GLAdomain can include a substitution of an aspartic acid residue at residue33. Substitution of a glutamine residue at residue 11 is particularlyuseful. For example, a glutamine residue at residue 11 and a glutamicacid residue at residue 33 or a phenylalanine at residue 29 may besubstituted. The GLA domain can include, for example, a substitution ofa glutamic acid or a phenylalanine residue at residue 29 and further caninclude a substitution of a glutamic acid or an aspartic acid at residue33. Such a polypeptide further can include a glutamic acid or anaspartic acid residue at amino acid 33.

Isolated nucleic acid molecules that include a nucleic acid sequenceencoding modified vitamin K-dependent polypeptides also are described.The nucleic acid molecules encode vitamin K-dependent polypeptides thatinclude a modified GLA domain that enhances membrane binding affinity ofthe polypeptide relative to a corresponding native vitamin K-dependentpolypeptide. The modified GLA domain of such encoded polypeptides caninclude a substitution at amino acid 11, 29, or 33 as discussed above.The native vitamin K-dependent polypeptide can be, for example, factorVII, factor VIIa, active-site modified factor VIIa, factor IX, or factorIXa.

The invention also features a mammalian host cell that includes avitamin k-dependent polypeptide. The polypeptide includes a modified GLAdomain that enhances membrane binding affinity of the polypeptiderelative to a corresponding native vitamin k-dependent polypeptide. Themodified GLA domain includes at least one amino acid substitution at,for example, amino acid 11, 12, 29, 33 or 34. The vitamin k-dependentpolypeptide may be, for example, factor VII or factor VIIa.

The invention also relates to a pharmaceutical composition that includesa pharmaceutically acceptable carrier and an amount of a vitamink-dependent polypeptide effective to inhibit clot formation in a mammal.The vitamin k-dependent polypeptide includes a modified GLA domain thatenhances membrane binding affinity of the polypeptide relative to acorresponding native vitamin k-dependent polypeptide. The modified GLAdomain includes at least one amino acid substitution. The vitamink-dependent polypeptide may be, for example, protein C, activatedprotein C or active site modified factor VIIa.

The invention also features a pharmaceutical composition that includes apharmaceutically acceptable carrier and an amount of a vitamink-dependent polypeptide effective to increase clot formation in amammal. The vitamin k-dependent polypeptide includes a modified GLAdomain that enhances membrane binding affinity of the polypeptiderelative to a corresponding native vitamin k-dependent polypeptide. Themodified GLA domain includes at least one amino acid substitution. Forexample, residue 11, 29, or 33 can be modified as discussed above. Thevitamin k-dependent polypeptide may be, for example, factor VII, factorVIIa, factor IX or factor IXa. The pharmaceutical composition also mayinclude soluble tissue factor. Such pharmaceutical compositions can beused to treat a bleeding disorder in a patient by administering thepharmaceutical composition to the patient.

A method of decreasing clot formation in a mammal is also described. Themethod includes administering an amount of a vitamin k-dependentpolypeptide effective to decrease clot formation in the mammal. Thevitamin k-dependent polypeptide includes a modified GLA domain thatenhances membrane binding affinity of the polypeptide relative to acorresponding native vitamin k-dependent polypeptide. The modified GLAdomain includes at least one amino acid substitution. The vitamink-dependent polypeptide may be, for example, protein C, activatedprotein C or active site modified factor VIIa.

The invention also features a method of increasing clot formation in amammal. The method includes administering an amount of a vitamink-dependent polypeptide effective to increase clot formation in themammal. The vitamin k-dependent polypeptide includes a modified GLAdomain that enhances membrane binding affinity of the polypeptiderelative to a corresponding native vitamin k-dependent polypeptide. Themodified GLA domain includes at least one amino acid substitution. Forexample, the modified GLA domain can include an amino acid substitutionat residue 11, 29, or 33, as discussed above. The vitamin k-dependentpolypeptide may be, for example, factor VII, factor VIIa, factor IX orfactor IXa.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the binding, with standard deviations, of wild type VIIa(open circles), VIIQ11E33 (filled circles), and bovine factor X (filledtriangles) to membranes.

FIG. 2 depicts the autoactivation of VIIQ11E33. The dashed line showsactivity in the absence of phospholipid.

FIG. 3 depicts the activation of factor X by factor VIIa. Results forwild type factor VIIa (open circles) and VIIaQ11E33 (filled circles) aregiven for a concentration of 0.06 nM.

FIG. 4 depicts the coagulation of human plasma by VIIa and VIIaQ11E33with soluble tissue factor.

FIG. 5 depicts the coagulation of plasma by factor VII zymogens andnormal tissue factor.

FIG. 6 depicts the inhibition of clot formation by active-site modifiedfactor VIIaQ11E33 (DEGR-VIIaQ11E33).

FIG. 7 depicts the circulatory time of factor VIIQ11E33 in rats.

FIG. 8 depicts the membrane interaction by normal and modified proteins.Panel A shows the interaction of wild type bovine protein C (opencircles) and bovine protein C-H11 (filled circles) with vesicles. PanelB shows the interaction of wild type human protein C (open circles) andhuman protein C-P11 (filled circles) with membranes. In both cases, thedashed line indicates the result if all of the added protein were boundto the membrane.

FIG. 9 depicts the influence of activated protein C on clotting times.In panel A, the average and standard deviation for three determinationsof clotting times for bovine plasma are shown for wild type bovine APC(open circles) and for bAPC-H11 (filled circles). In panel B, theaverage and standard deviation of three replicates of human plasmacoagulation for the wild type human (open circles) and human APC-P11(filled circles) are shown.

FIG. 10 depicts the inactivation of factor Va by bovine and human APC.Panel A depicts the inactivation of factor Va by wild type bovine APC(open circles) and bovine APC-H11 (filled circles). Panel B depicts theinactivation of human factor Va in protein S-deficient plasma by eitherwild type human APC (open circles) and human APC-H11 (filled circles).

FIG. 11 depicts the electrostatic distribution of protein Z. Verticallines denote electropositive regions and horizontal lines denoteelectronegative regions

FIG. 12 depicts the membrane binding and activity of various protein Cs.Panel A shows membrane binding by wild type protein C (open circles),the P11H mutant of protein C (filled squares), Q33E,N34D mutant (filledcircles) and bovine prothrombin (open squares). Panel B shows inhibitionof blood coagulation by these mutants. Panel C shows the inactivation offactor Va.

FIG. 13 compares membrane binding and activity of human protein Cmutants. Panel A compares the membrane binding of wild-type (opencircles), E33 (open triangles) and E33D34 (filled circles). Panel Bcompares the coagulation times using wild-type (open triangles), E33(open circles) and E33D34 (filled circles).

FIG. 14 compares membrane binding (Panel A) and coagulation inhibition(Panel B) with wild-type (open squares), H11 (filled circles), E33D34(open triangles) and the triple H11E33D34 mutant (open circles) ofbovine protein C.

FIG. 15 depicts the membrane interaction properties of different vitaminK-dependent proteins. Panel A compares membrane interaction of human(filled circles) and bovine (open circles) factor X. Panel B showsmembrane interaction by normal bovine prothrombin fragment 1 (opencircles), fragment 1 modified with TNBS in the absence of calcium(filled circles) and fragment 1 modified with TNBS in the presence of 25mM calcium (filled squares). Panel C shows the rate of protein Z bindingto vesicles at pH 9 (filled circles) and 7.5 (open circles).

DETAILED DESCRIPTION

In one aspect, the invention features a vitamin k-dependent polypeptideincluding a modified GLA domain with enhanced membrane binding affinityrelative to a corresponding native vitamin k-dependent polypeptide.Vitamin k-dependent polypeptides are a group of proteins that utilizevitamin k in their biosynthetic pathway to carboxylate the side chainsof glutamic acid residues in protein precursors. The GLA domain contains9-13 γ-carboxyglutamic acid residues in the N-terminal region of thepolypeptide, typically from amino acid 1 to about amino acid 45. ProteinZ, protein S, factor X, factor II (prothrombin), factor IX, protein C,factor VII and Gas6 are examples of vitamin k-dependent polypeptides.Amino acid positions of the polypeptides discussed herein are numberedaccording to factor IX. Protein S, protein C, factor X, factor VII andhuman prothrombin all have one less amino acid (position 4) and must beadjusted accordingly. For example, actual position 10 of bovine proteinC is a proline, but is numbered herein as amino acid 11 for ease ofcomparison throughout. As used herein, the term “polypeptide” is anychain of amino acids, regardless of length or post-translationalmodification. Amino acids have been designated herein by standard threeletter and one letter abbreviations.

Modifications of the GLA domain include at least one amino acidsubstitution. The substitutions may be conservative or non-conservative.Conservative amino acid substitutions replace an amino acid with anamino acid of the same class, whereas non-conservative amino acidsubstitutions replace an amino acid with an amino acid of a differentclass. Non-conservative substitutions may result in a substantial changein the hydrophobicity of the polypeptide or in the bulk of a residueside chain. In addition, non-conservative substitutions may make asubstantial change in the charge of the polypeptide, such as reducingelectropositive charges or introducing electronegative charges. Examplesof non-conservative substitutions include a basic amino acid for anon-polar amino acid, or a polar amino acid for an acidic amino acid.The amino acid substitution may be at amino acid 11, 12, 29, 33, or 34.Preferably, the amino acid substitution is at amino acid 11, 33, or 34.The modified GLA domain may include an amino acid sequence which, in thecalcium saturated state, contributes to formation of a tertiarystructure having a cationic core with a halo of electronegative charge.Without being bound by a particular theory, enhanced membrane affinitymay result from a particular electrostatic pattern consisting of anelectropositive core completely surrounded by an electronegativesurface.

Many vitamin K-dependent polypeptides are substrates for membrane-boundenzymes. Since no vitamin K-dependent polypeptides display the maximumpotential membrane-binding affinity of a GLA domain, all must containamino acids whose purpose is to reduce binding affinity. Consequently,many vitamin K-dependent polypeptides contain amino acids that arenon-optimal from the standpoint of maximum affinity. These residueseffectively disrupt the binding site to provide a more rapid turnoverfor an enzymatic reaction.

Lowered membrane affinity may serve several purposes. High affinity isaccompanied by slow exchange, which may limit reaction rates. Forexample, when the prothrombinase enzyme is assembled on membranes withhigh affinity for substrate, protein exchange from the membrane, ratherthan enzyme catalysis, is the limiting. Lu, Y. and Nelsestuen, G. L.,1996, Biochemistry, 35:8201-8209. Alternatively, adjustment of membraneaffinity by substitution with non-optimum amino acids may balance thecompeting processes of procoagulation (factor X, IX, VII andprothrombin) and anticoagulation (protein C, S). Although membraneaffinities of native proteins may be optimal for normal states,enhancement of membrane affinity can produce proteins that are usefulfor in vitro study as well as improved therapeutics for regulating bloodclotting in pathological conditions in vivo.

Various examples of GLA domain modified vitamin k-dependent polypeptidesare described below.

The vitamin k-dependent polypeptide may be protein C or activatedprotein C (APC). Amino acid sequences of the wild-type human (hC) andbovine (bC) protein C GLA domain are shown in Table 1. X is a Gla or Gluresidue. In general, a protein with neutral (e.g., Q) or anionicresidues (e.g., D, E) at positions 11, 33 and 34 will have highermembrane affinity.

TABLE 1 hC: ANS-FLXXLRH₁₁SSLXRXCIXX₂₁ICDFXXAKXI₃₁FQNVDDTLAF₄₁WSKH (SEQID NO:1) bC: ANS-FLXXLRP₁₁GNVXRXCSXX₂₁VCXFXXARXI₃₁FQNTXDTMAF₄₁WSFY (SEQID NO:2)

The modified GLA domain of protein C or APC may include, for example, aglutamic acid residue at amino acid 33 and an aspartic acid residue atamino acid 34. The glutamic acid at position 33 may be further modifiedto γ-carboxyglutamic acid in vivo. For optimum activity, the modifiedGLA domain may include an additional substitution at amino acid 11. Forexample, a glutamine residue may be substituted at amino acid 11 oralternatively, a glutamic acid or an aspartic acid residue may besubstituted. A histidine residue may be substituted at amino acid 11 inbovine protein C. A further modification can include a substitution atamino acid 12 of a glycine residue for serine. Replacement of amino acid29 by phenylalanine, the amino acid found in prothrombin, is anotheruseful modification. Modified protein C with enhanced membrane bindingaffinity may be used in place of other injectable anticoagulants such asheparin. Heparin is typically used in most types of surgery, but suffersfrom a low efficacy/toxicity ratio. In addition, modified protein C withenhanced membrane affinity may be used in place of oral anticoagulantsin the coumarin family, such as warfarin.

These modifications can also be made with active site modified APC. Theactive site of APC may be inactivated chemically, for example, byN-dansyl-glutamyl glycylarginylchloromethylketone (DEGR) or bysite-directed mutagenesis of the active site. Sorensen, B. B. et al.,1997, J. Biol. Chem., 272:11863-11868. Active site-modified APCfunctions as an inhibitor of the prothrombinase complex. Enhancedmembrane affinity of active site modified APC may result in a moretherapeutically effective polypeptide.

The vitamin k-dependent polypeptide may be factor VII or the active formof factor VII, factor VIIa. Native or naturally-occurring factor VIIpolypeptide has low affinity for membranes. Amino acid sequences of thewild-type human (hVII) and bovine (bVII) factor VII GLA domain are shownin Table 2.

TABLE 2 hVII: ANA-FLXXLRP₁₁GSLXRXCKXX₂₁QCSFXXARXI₃₁FKDAXRTKLF₄₁WISY (SEQID NO:3) bVII: ANG-FLXXLRP₁₁GSLXRXCRXX₂₁LCSFXXAHXI₃₁FRNXXRTRQF₄₁WVSY(SEQ ID NO:4)

The GLA domain of factor VII or VIIa can contain a substitution, forexample, at amino acid 11, 12, 29, or 33. The modified GLA domain offactor VII or factor VIIa may include, for example, a glutamic acid, aglutamine, an asparagine, or an aspartic acid residue at amino acid 11,a phenylalanine or a glutamic acid residue at amino acid 29, or anaspartic acid or a glutamic acid residue at amino acid 33. The modifiedGLA domain can include combinations of such substitutions at amino acidresidues 11 and 29, at residues 11 and 33, at residues 11, 29 and 33, orat residues 29 and 33. For example, the GLA domain of factor VII orfactor VIIa may include a glutamine residue at amino acid 11 and aglutamic acid residue at amino acid 33, or a glutamine residue at aminoacid 11 and a phenylalanine residue at amino acid 29. Vitamink-dependent polypeptide modified in this manner has a much higheraffinity for membranes than the native or wild type polypeptide. It alsohas a much higher activity in autoactivation, in factor Xa generationand in several blood clotting assays. Activity is particularly enhancedat marginal coagulation conditions, such as low levels of tissue factorand/or phospholipid. For example, modified factor VII is about 4 timesas effective as native VIIa at optimum thromboplastin levels, but isabout 20-fold as effective at 1% of optimum thromboplastin levels.Marginal pro-coagulation signals are probably most predominant in vivo.Presently available clotting assays that use optimum levels ofthromboplastin cannot detect clotting time differences between normalplasma and those from hemophilia patients. Clotting differences betweensuch samples are only detected when non-optimal levels of thromboplastinor dilute thromboplastin are used in clotting assays.

Another example of a vitamin k-dependent polypeptide is active-sitemodified Factor VIIa. The active site of factor VIIa may be modifiedchemically, for example, by DEGR or by site-directed mutagenesis of theactive site. DEGR-modified factor VII is an effective inhibitor ofcoagulation by several routes of administration. Arnljots, B. et al.,1997, J. Vasc. Surg., 25:341-346. Modifications of the GLA domain maymake active-site modified Factor VIIa more efficacious due to highermembrane affinity. The modified GLA domain of active-site modifiedFactor VIIa may include substitutions as described above for Factor VII.For example, a glutamine residue at amino acid 11 and a glutamic acidresidue at amino acid 33.

The vitamin K-dependent polypeptide may also be Factor IX or the activeform of Factor IX, Factor IXa. As with active site-modified factor VIIa,active site modified IXa and Xa may be inhibitors of coagulation. Aminoacid sequences of the wild-type human (hIX) and bovine (bIX) factor IXGLA domain are shown in Table 3. Suitable substitutions for factor IXare described above. For example, substitutions can include anasparagine, an aspartic acid or glutamic acid residue at amino acid 11,a phenylalanine or glutamic acid residue at amino acid 29, a glutamineor aspartic acid residue at amino acid 33, or an aspartic acid residueat amino acid 34.

TABLE 3 hIX: YNSGKLXXFVQ₁₁GNLXRXCMXX₂₁KCSFXXARXV₃₁FXNTXRTTXF₄₁WKQY (SEQID NO:5) bIX: YNSGKLXXFVQ₁₁GNLXRXCMXX₂₁KCSFXXARXV₃₁FXNTXKRTTXF₄₁WKQY(SEQ ID NO:6)

In another aspect, the invention features a mammalian host cellincluding a vitamin k-dependent polypeptide having a modified GLA domainthat enhances membrane binding affinity of the polypeptide relative to acorresponding native vitamin k-dependent polypeptide. The modified GLAdomain includes at least one amino acid substitution as discussed above.The mammalian host cell may include, for example, modified factor VII ormodified factor VIIa, a discussed above. The GLA domain of modifiedfactor VII or modified factor VIIa may contain, for example, an aminoacid substitution at amino acid 11 and at amino acid 33. Preferably, theamino acid substitution includes a glutamine residue at amino acid 11and a glutamic acid residue at amino acid 33.

Suitable mammalian host cells are able to modify vitamin k-dependentpolypeptide glutamate residues to γ-carboxyglutamate. Mammalian cellsderived from kidney and liver are especially useful as host cells.

Nucleic Acids Encoding Modified Vitamin K-dependent Polypeptides

Isolated nucleic acid molecules encoding modified vitamin K-dependentpolypeptides of the invention can be produced by standard techniques. Asused herein, “isolated” refers to a sequence corresponding to part orall of a gene encoding a modified vitamin K-dependent polypeptide, butfree of sequences that normally flank one or both sides of the wild-typegene in a mammalian genome. An isolated polynucleotide can be, forexample, a recombinant DNA molecule, provided one of the nucleic acidsequences normally found immediately flanking that recombinant DNAmolecule in a naturally-occurring genome is removed or absent. Thus,isolated polynucleotides include, without limitation, a recombinant DNAthat exists as a separate molecule (e.g., a cDNA or genomic DNA fragmentproduced by PCR or restriction endonuclease treatment) independent ofother sequences as well as recombinant DNA that is incorporated into avector, an autonomously replicating plasmid, a virus (e.g., aretrovirus, adenovirus, or herpes virus), or into the genomic DNA of aprokaryote or eukaryote. In addition, an isolated polynucleotide caninclude a recombinant DNA molecule that is part of a hybrid or fusionpolynucleotide.

It will be apparent to those of skill in the art that a polynucleotideexisting among hundreds to millions of other polynucleotides within, forexample, cDNA or genomic libraries, or gel slices containing a genomicDNA restriction digest is not to be considered an isolatedpolynucleotide.

Isolated nucleic acid molecules are at least about 14 nucleotides inlength. For example, the nucleic acid molecule can be about 14 to 20,20-50, 50-100, or greater than 150 nucleotides in length. In someembodiments, the isolated nucleic acid molecules encode a full-lengthmodified vitamin K-dependent polypeptide. Nucleic acid molecules can beDNA or RNA, linear or circular, and in sense or antisense orientation.

Specific point changes can be introduced into the nucleic acid sequenceencoding wild-type vitamin K-dependent polypeptides by, for example,oligonucleotide-directed mutagenesis. In this method, a desired changeis incorporated into an oligonucleotide, which then is hybridized to thewild-type nucleic acid. The oligonucleotide is extended with a DNApolymerase, creating a heteroduplex that contains a mismatch at theintroduced point change, and a single-stranded nick at the 5′ end, whichis sealed by a DNA ligase. The mismatch is repaired upon transformationof E. coli, and the gene encoding the modified vitamin K-dependentpolypeptide can be re-isolated from E. coli. Kits for introducingsite-directed mutations can be purchased commercially. For example,Muta-Gene® in-vitro mutagenesis kits can be purchased from Bio-RadLaboratories, Inc. (Hercules, Calif.).

Polymerase chain reaction (PCR) techniques also can be used to introducemutations. See, for example, Vallette et al., Nucleic Acids Res., 1989,17(2):723-733. PCR refers to a procedure or technique in which targetnucleic acids are amplified. Sequence information from the ends of theregion of interest or beyond typically is employed to designoligonucleotide primers that are identical in sequence to oppositestrands of the template to be amplified, whereas for introduction ofmutations, oligonucleotides that incorporate the desired change are usedto amplify the nucleic acid sequence of interest. PCR can be used toamplify specific sequences from DNA as well as RNA, including sequencesfrom total genomic DNA or total cellular RNA. Primers are typically 14to 40 nucleotides in length, but can range from 10 nucleotides tohundreds of nucleotides in length. General PCR techniques are described,for example in PCR Primer: A Laboratory Manual, Ed. by Dieffenbach, C.and Dveksler, G., Cold Spring Harbor Laboratory Press, 1995.

Nucleic acids encoding modified vitamin K-dependent polypeptides alsocan be produced by chemical synthesis, either as a single nucleic acidmolecule or as a series of oligonucleotides. For example, one or morepairs of long oligonucleotides (e.g., >100 nucleotides) can besynthesized that contain the desired sequence, with each pair containinga short segment of complementarity (e.g., about 15 nucleotides) suchthat a duplex is formed when the oligonucleotide pair is annealed. DNApolymerase is used to extend the oligonucleotides, resulting in adouble-stranded nucleic acid molecule per oligonucleotide pair, whichthen can be ligated into a vector.

Production Of Modified Vitamin K-dependent Polypeptides

Modified vitamin K-dependent polypeptides of the invention can beproduced by ligating a nucleic acid sequence encoding the polypeptideinto a nucleic acid construct such as an expression vector, andtransforming a bacterial or eukaryotic host cell with the expressionvector. In general, nucleic acid constructs include a regulatorysequence operably linked to a nucleic acid sequence encoding a vitaminK-dependent polypeptide. Regulatory sequences do not typically encode agene product, but instead affect the expression of the nucleic acidsequence. As used herein, “operably linked” refers to connection of theregulatory sequences to the nucleic acid sequence in such a way as topermit expression of the nucleic acid sequence. Regulatory elements caninclude, for example, promoter sequences, enhancer sequences, responseelements, or inducible elements.

In bacterial systems, a strain of Escherichia coli such as BL-21 can beused. Suitable E. coli vectors include without limitation the pGEXseries of vectors that produce fusion proteins with glutathioneS-transferase (GST). Transformed E. coli are typically grownexponentially, then stimulated with isopropylthiogalactopyranoside(IPTG) prior to harvesting. In general, such fusion proteins are solubleand can be purified easily from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. The pGEX vectors are designed to include thrombin or factorXa protease cleavage sites such that the cloned target gene product canbe released from the GST moiety.

In eukaryotic host cells, a number of viral-based expression systems canbe utilized to express modified vitamin K-dependent polypeptides. Anucleic acid encoding vitamin K-dependent polypeptide can be clonedinto, for example, a baculoviral vector such as pBlueBac (Invitrogen,San Diego, Calif.) and then used to co-transfect insect cells such asSpodoptera frugiperda (Sf9) cells with wild-type DNA from Autographacalifornica multiply enveloped nuclear polyhedrosis virus (AcMNPV).Recombinant viruses producing the modified vitamin K-dependentpolypeptides can be identified by standard methodology. Alternatively, anucleic acid encoding a vitamin K-dependent polypeptide can beintroduced into a SV40, retroviral, or vaccinia based viral vector andused to infect host cells.

Mammalian cell lines that stably express modified vitamin K-dependentpolypeptides can be produced by using expression vectors with theappropriate control elements and a selectable marker. For example, theeukaryotic expression vector pCDNA.3.1⁺ (Invitrogen, San Diego, Calif.)is suitable for expression of modified vitamin K-dependent polypeptidesin, for example, COS cells, HEK293 cells, or baby hamster kidney cells.Following introduction of the expression vector by electroporation, DEAEdextran-, calcium phosphate-, liposome-mediated transfection, or othersuitable method, stable cell lines can be selected. Alternatively,transiently transfected cell lines are used to produce modified vitaminK-dependent polypeptides. Modified vitamin K-dependent polypeptides alsocan be transcribed and translated in vitro using wheat germ extract orrabbit reticulocyte lysate.

Modified vitamin K-dependent polypeptides can be purified fromconditioned cell medium by applying the medium to an immunoaffinitycolumn. For example, an antibody having specific binding affinity forFactor VII can be used to purify modified Factor VII. Alternatively,concanavalin A (Con A) chromatography and anion-exchange chromatography(e.g., DEAE) can be used in conjunction with affinity chromatography topurify factor VII. Calcium dependent or independent monoclonalantibodies that have specific binding affinity for factor VII can beused in the purification of Factor VII.

Modified vitamin K-dependent polypeptides such as modified protein C canbe purified by anion-exchange chromatography, followed by immunoaffinitychromatography using an antibody having specific binding affinity forprotein C.

Modified vitamin K-dependent polypeptides also can be chemicallysynthesized using standard techniques. See, Muir, T. W. and Kent, S. B.,Curr. Opin. Biotechnol., 1993, 4(4):420-427, for a review of proteinsynthesis techniques.

Pharmaceutical Compositions

The invention also features a pharmaceutical composition including apharmaceutically acceptable carrier and an amount of a vitamink-dependent polypeptide effective to inhibit clot formation in a mammal.The vitamin k-dependent polypeptide includes a modified GLA domain withat least one amino acid substitution that enhances membrane bindingaffinity of the polypeptide relative to a corresponding native vitamink-dependent polypeptide. Useful modified vitamin k-dependentpolypeptides of the pharmaceutical compositions can include, withoutlimitation, protein C or APC, active-site modified APC, active-sitemodified factor VIIa, active-site modified factor IXa, and active-sitemodified factor Xa as discussed above.

The concentration of a vitamin k-dependent polypeptide effective toinhibit clot formation in a mammal may vary, depending on a number offactors, including the preferred dosage of the compound to beadministered, the chemical characteristics of the compounds employed,the formulation of the compound excipients and the route ofadministration. The optimal dosage of a pharmaceutical composition to beadministered may also depend on such variables as the overall healthstatus of the particular patient and the relative biological efficacy ofthe compound selected. These pharmaceutical compositions may be used toregulate coagulation in vivo. For example, the compositions may be usedgenerally for the treatment of thrombosis. Altering only a few aminoacid residues of the polypeptide as described above, generally does notsignificantly affect the antigenicity of the mutant polypeptides.

Vitamin k-dependent polypeptides that include modified GLA domains maybe formulated into pharmaceutical compositions by admixture withpharmaceutically acceptable non-toxic excipients or carriers. Suchcompounds and compositions may be prepared for parenteraladministration, particularly in the form of liquid solutions orsuspensions in aqueous physiological buffer solutions; for oraladministration, particularly in the form of tablets or capsules; or forintranasal administration, particularly in the form of powders, nasaldrops, or aerosols. Compositions for other routes of administration maybe prepared as desired using standard methods.

Formulations for parenteral administration may contain as commonexcipients sterile water or saline, polyalkylene glycols such aspolyethylene glycol, oils of vegetable origin, hydrogenatednaphthalenes, and the like. In particular, biocompatible, biodegradablelactide polymer, lactide/glycolide copolymer, orpolyoxethylene-polyoxypropylene copolymers are examples of excipientsfor controlling the release of a compound of the invention in vivo.Other suitable parenteral delivery systems include ethylene-vinylacetate copolymer particles, osmotic pumps, implantable infusionsystems, and liposomes. Formulations for inhalation administration maycontain excipients such as lactose, if desired. Inhalation formulationsmay be aqueous solutions containing, for example,polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or theymay be oily solutions for administration in the form of nasal drops. Ifdesired, the compounds can be formulated as gels to be appliedintranasally. Formulations for parenteral administration may alsoinclude glycocholate for buccal administration

In an alternative embodiment, the invention also features apharmaceutical composition including a pharmaceutically acceptablecarrier and an amount of a vitamin k-dependent polypeptide effective toincrease clot formation in a mammal. The vitamin k-dependent polypeptideincludes a modified GLA domain with at least one amino acid substitutionthat enhances membrane binding affinity of the polypeptide relative to acorresponding native vitamin k-dependent polypeptide. Thesepharmaceutical compositions may be useful for the treatment of clottingdisorders such as hemophilia A, hemophilia B and liver disease.

In this embodiment, useful vitamin k-dependent polypeptides of thepharmaceutical compositions can include, without limitations, Factor VIIor the active form of Factor VII, Factor VIIa. The modified GLA domainof Factor VII or Factor VIIa may include substitutions at amino acid 11and amino acid 33, for example, a glutamine residue at amino acid 11 anda glutamic acid residue at amino acid 33. The pharmaceutical compositionmay further comprise soluble tissue factor. Factor VII is especiallycritical to blood coagulation because of its location at the initiationof the clotting cascade, and its ability to activate two proteins,factors IX and X. Direct activation of factor X by factor VIIa isimportant for possible treatment of the major forms of hemophilia, typesA and B, since the steps involving factors IX and VIII are bypassedentirely. Administration of factor VII to patients has been found to beefficacious for treatment of some forms of hemophilia. Improvement ofthe membrane affinity of factor VII or VIIa by modification of the GLAdomain provides the potential to make the polypeptide more responsive tomany coagulation conditions, to lower the dosages of VII/VIIa needed, toextend the intervals at which factor VII/VIIa must be administered, andto provide additional qualitative changes that result in more effectivetreatment. Overall, improvement of the membrane contact site of factorVII may increase both its activation rate as well as improve theactivity of factor VIIa on factor X or IX. These steps may have amultiplicative effect on overall blood clotting rates in vivo, resultingin a very potent factor VIIa for superior treatment of several bloodclotting disorders.

Other useful vitamin k-dependent polypeptides for increasing clotformation include Factor IX and Factor IXa.

In another aspect, methods for decreasing clot formation in a mammal aredescribed. The method includes administering an amount of vitamink-dependent polypeptide effective to decrease clot formation in themammal. The vitamin k-dependent polypeptide includes a modified GLAdomain that enhances membrane binding affinity of the polypeptiderelative to a corresponding native vitamin k-dependent polypeptide. Themodified GLA domain includes at least one amino acid substitution.Modified protein C or APC or modified active-site blocked factors VIIa,IXa, Xa and APC may be used for this method.

In another aspect, the invention also features methods for increasingclot formation in a mammal that includes administering an amount ofvitamin k-dependent polypeptide effective to increase clot formation inthe mammal. The vitamin k-dependent polypeptide includes a modified GLAdomain that enhances membrane binding affinity of the polypeptiderelative to a corresponding native vitamin k-dependent polypeptide. Themodified GLA domain includes at least one amino acid substitution.Modified factor VII or VIIa and modified factor IX or IXa may be used inthis method.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Factor VII with Enhanced Membrane Affinity andActivity

It has been found that the membrane binding affinity of human bloodclotting factor VII can be increased by site-directed mutagenesis. Theproperties of a P11Q,K33E mutant (referred to herein as Factor VIIQ11E33or mutant factor VII) have been characterized. Membrane affinity wasincreased over wild type protein by about 20-fold. Autoactivation by themutant was increased by at least 100-fold over that of wild type factorVII. The activated form of VIIQ11E33 (referred to as VIIaQ11E33)displayed about 10-fold higher activity toward factor X. The coagulationactivity of VIIaQ11E33 with soluble tissue factor in normal plasma wasabout 10-fold higher than that of wild type VIIa. Coagulation activityof the zymogen, VIIQ11E33, with normal tissue factor (supplied as a1:100 dilution of thromboplastin-HS), was 20-fold higher than wild typeFactor VII. The degree to which activity was enhanced was dependent onconditions, with VIIQ11E33 being especially active under conditions oflow coagulation stimuli.

In general, protein concentrations were determined by the Bradford assayusing bovine serum albumin as the standard. Bradford, M. M., 1976,Analyt. Biochem. 248-254. Molar concentrations were obtained from themolecular weights of 50,000 for factor VII and 55,000 for factor X.Unless indicated, all activity measurements were conducted in standardbuffer (0.05 M Tris, pH 7.5, 100 mM NaCl).

Production of Mutant Factor VII: Mutant factor VII was generated fromwild type factor VII cDNA (GenBank Accession number M13232, NIDg182799). Petersen et al., 1990, Biochemistry 29:3451-3457. The P11Qmutation (change of amino acid 11 from a proline residue to a glutamineresidue) and the K33E mutation (change of amino acid 33 from a lysineresidue to a glutamic acid residue) were introduced into the wild typefactor VII cDNA by a polymerase chain reaction strategy essentially asdescribed by Vallette et al., 1989, Nucleic Acids Res. 17:723-733.During this process, a mutation-diagnostic XmaIII restriction enzymesite was eliminated. Four PCR primers were designed to prime synthesisof two mutant fragments of M13232, one from MluI to BglII, positions 221to 301, and the other from BglII to SstII, positions 302 to 787. Theseprimers were used under standard PCR cycling conditions (GENEAMP, PerkinElmer) to prime fragment synthesis using 1 ng of the wild-type factorVII cDNA as template. The resulting fragments were gel purified anddigested with MluII and BglII or BglII and SstII. The two purifiedfragments were then ligated into the factor VIII cDNA in the expressionvector Zem219b from which the corresponding wild-type sequence had beenremoved as a MluI-SstII fragment. Petersen et al., 1990 supra. Themutated fragments were sequenced in their entirety to confirm the P11Qand K33E substitutions, as well as to eliminate the possibility of otherPCR-induced sequence changes.

Transfection, Selection and Purification: Baby hamster kidney (BHK)cells were grown in Dubeccos modified Eagles medium supplemented with10% fetal calf serum and penicillin-streptomycin. Subconfluent cellswere transfected with the factor VII expression plasmid usinglipofectAMINE (Gibco BRL) according to the manufacturersrecommendations. Two days post-transfection, cells were trypsinized anddiluted to selective medium containing 1 μM methotrexate (MTX).Stably-transfected BHK cells were subsequently cultured in serum-freeDubeccos modified Eagles medium supplemented withpenicillin-streptomycin, 5 μg/mL vitamin K₁ and 1 μM MTX, andconditioned medium was collected. The conditioned medium was appliedtwice to an immunoaffinity column composed of a calcium-dependentmonoclonal antibody (CaFVII22) coupled to Affi-Gel 10. Nakagaki et al.,1991, Biochemistry, 30:10819-10824. The final purified Factor VIIQ11E33ran as a single band on SDS polyacrylamide gel electrophoresis, with noevidence of factor VIIa in the preparation. The pure VII(P11Q,K33E)mutant showed 1400-2800 factor VII units/mg.

Activation of Factor VII: Activated Factor VIIaQ11E33 was formed bybovine factor Xa cleavage of VIIQ11E33 (1:100 weight ratio, incubationfor 1 hr at 37° C.). Alternatively, Factor VIIaQ11E33 was obtained byautoactivation (37° C., 20 min) in a mixture containing 7 μM VIIQ11E33,0.7 μM sTF and phospholipid (phosphatidylserine/phosphatidylcholine(PS/PC), 25/75, 0.1 g/g protein).

Wild-type factor VIIa was a homogeneous, recombinant protein (NOVONordisk). Two preparations consisted of a commercial, lyophilizedproduct and non-lyophilized product. The latter protein was furtherpurified on FPLC mono-Q and showed a specific activity of 80,000units/mg, calibrated with a George King NPP standard.

Enhanced membrane interaction by Factor VIIQ11E33: Phospholipidpreparation, assay and measurement of protein-membrane binding wasconducted by the method described by Nelsestuen and Lim, 1977,Biochemistry, 30:10819-10824. Large unilamellar vesicles (LUVs) andsmall unilamellar vesicles (SUVs) were prepared by methods describedpreviously. Hope, M. J., et al., Biochem. Biophys. Acta., 812:55-65;Huang, C., 1969, Biochemistry, 8:344-352. Highly pure phosphatidylserine(bovine brain) and egg phosphatidylcholine (Sigma Chemical Co.) weremixed in chloroform. The solvent was removed by a stream of nitrogengas. The dried phospholipids were suspended in buffer. SUVs were formedby sonication and gel filtration while LUVs were formed by freeze-thawand extrusion. Phospholipid concentrations were determined by organicphosphate assay assuming a phosphorous:phospholipid weight ratio of 25.

SUVS of either PS/PC (25/75) or PS/PC (10/90) were prepared. Protein wasadded to phospholipid at the weight ratios shown in FIG. 1.Protein-membrane binding was assayed by light scattering at 90° by themethod of Nelsestuen and Lim, 1977 supra. Briefly, the light scatteringintensity of phospholipid vesicles alone (I₁) and after addition ofprotein (I₂) were measured and corrected for background from buffer andunbound protein. The molecular weight ratio of the protein-vesiclecomplex (M₂) to that of the vesicles alone (M1), can be estimated fromthe relationship in equation 1, where δn/δc is the refractive index ofthe respective species.

I ₂ /I ₁=(M ₂ /M ₁)²(δn/δc ₂ /δn/δc ₁)²  (eq. 1)

If phospholipid and protein concentrations are known, the concentrationof bound [P*PL] and free protein [P] can be estimated. These values,together with the maximum protein binding capacity [P*PL_(max)] of thevesicles (assumed to be 1.0 g/g for all proteins) can be used to obtainthe equilibrium constant for protein-membrane interaction by therelationship in equation 2, where all concentrations are expressed asmolar protein or protein binding sites.

K _(D) =[P][P*PL _(max) −P*PL]/[P*PL]  (eq. 2)

Binding was assessed at 5 mM calcium and is expressed as the ratio,M2/M1.

FIG. 1 shows the binding of wild type VIIa (open circles) and factorVIIQ11E33 (filled circles) to membranes of either PS/PC=25/75, 25 μg/ml(FIG. 1A) or PS/PC=10/90, 25 μg/ml (FIG. 1B). VIIQ11E33 had much higheraffinity than wild type protein. Binding to PS/PC (25/75) was at thequantitative level so that [Protein_(free)] was essentially zero.Consequently, Kd values could not be estimated from this data. Membranebinding of bovine factor X (filled triangles) is shown in FIG. 1 as areference. Bovine factor X is one of the highest affinity proteins inthis family, giving a Kd for PS/PC (20/80) at 2 mM calcium of 40 nM.McDonald et al., 1997, Biochemistry, 36:5120-5127. The Kd for bovinefactor X, obtained from the result at a protein/phospholipid ratio of0.55 (FIG. 1), was 0.025 μM.

Binding of wild-type and mutant Factor VII to membranes of PS/PC (10/90)was also determined (FIG. 1B). The VIIQ11E33 bound at less than thequantitative level, which allowed a binding constant to be estimatedfrom the relationship in equation 3.

Kd=[Protein_(free)][Binding sites_(free)]/[Protein_(bound)]  (eq. 3)

[Binding sites_(free)] were estimated from equation 4, assuming amaximum M2/M1 of 1.0 (i.e., [Bindingsites_(total)]=[Phospholipid_(weight conc.)/Protein_(MW)]). This is acommon value observed for several proteins of this family. See McDonaldet al., 1997, supra.

[Binding sites_(free)]=[Binding sites_(total)]−[Protein_(bound)]  (eq.4)

Using these assumptions and the data at a protein to phospholipid ratioof 0.37, Kd values were 0.7 μM for bovine factor X, 5.5 μM for wild typefactor VII and 0.23 μM for VIIQ11E33. Thus, it was clear that factorVIIQ11E33 was greatly improved in membrane binding affinity over wildtype factor VII and had one of the highest membrane-binding affinitiesamong the vitamin K-dependent proteins.

Enhanced activation of factor VIIQ11E33: The first step in coagulationinvolves the activation of factor VII. Autoactivation of VII wasconducted in a solution containing 100 nM sTF (highly purifiedrecombinant product from Dr. Walter Kisiel, Fiore et al., 1994, J. Biol.Chem., 269:143-149), 36 nM VIIQ11E33 and PS/PC (25/75, 22 μg/mL).Activity of VIIaQ11E33 was estimated at various time intervals byaddition of 0.15 mm substrate S-2288 (Kabi) and assessing the rate ofp-nitrophenylphosphate product release by absorbance change at 405 nm.Initial activity of the VIIQ11E33 preparation was less than 4% that offully active VIIaQ11E33.

VIIQ11E33 was found to be a much better substrate for activation thanwild-type factor VII. FIG. 2 shows autoactivation of factor VIIQ11E33.The data were analyzed by the relationship in equation 5 (equation 7 ofFiore et al., 1994, supra).

ln[VIIa] _(t) =ln[VIIa] ₀ +kcat*y*t  (eq. 5)

ln[VIIa]_(t) is the factor VIIa concentration at time t, kcat is thecatalytic rate constant for factor VIIa acting on VII and y is thefractional saturation of VIIa sites. For wild-type factor VIIa, thisrelationship and 1 μM sTF gave a kcat of 0.0045/s and a kcat/Km ratio of7*10³ M⁻¹s⁻¹. See, Fiore et al., 1994, supra. For the VIIQ11E33 enzyme,autoactivation was rapid (FIG. 2) and it was only possible to estimate alower limit for kcat. This was obtained from the VIIa doubling time ofabout 25 seconds (kcat=(ln2)/t_(½)). The resulting value(kcat_(min)=0.03/s), along with the substrate concentration of thisreaction (3.6*10⁻⁸ M) and the assumption that y=1.0, gave a value forkcat/[S]=8*10⁵ M⁻¹s⁻¹. This should be far below the true kcat/Km forVIIaQ11E33, but was about 100-times greater than the value of kcat/Kmfor wild type factor VIIa/sTF estimated by Fiore et al., 1994, supra.Thus, the combination of VIIaQ11E33 enzyme and Factor VIIQ11E33substrate was superior to wild type proteins in the activation step ofcoagulation. This suggested that VIIQ11E33 was superior to wild typeenzyme when coagulation conditions were minimal.

Enhanced activity of VIIaQ11E33: Once generated, factor VIIa activateseither factor X or factor IX. Activation of bovine factor X (0.1 μM) byfactor VIIa was carried out in 50 mM Tris.HCl buffer, pH 7.5 containing100 mM NaCl, 5 mM calcium, various amounts of phospholipid (PS/PC,25/75) and 1 mg/mL bovine serum albumin at 22.5° C. Factor VIIa (0.06 nMof VIIaQ11E33 or 0.6 nM wild type VIIa) was added at zero time and Xaactivity at 1, 3 and 5 minute time points was determined. Aliquots ofthe reaction mixture (0.2 mL) were mixed with buffer (0.2 mL) containing10 mM EDTA and 0.4 mM S-2222 (Kabi), a chromogenic substrate for factorXa. Absorbance change at 405 nm was determined in a Beckman DU8spectrophotometer. The amount of factor Xa generated was calculated fromthe extinction coefficient (1*10⁴ M⁻¹cm⁻¹) of the p-nitrophenylphosphatereaction product and a velocity of 33/sec for substrate hydrolysis bypurified bovine Xa under the conditions of this assay.

FIG. 3 compares the ability of wild type factor VIIa (open circles) andVIIaQ11E33 (closed circles) to activate factor X in a purified system.Again, VIIaQ11E33 was far superior to wild type factor VIIa in thisreaction. The difference was greatest at low phospholipid concentrationsand diminished to 2-fold at 200 μg phospholipid per mL. This wasexpected from the fact that high membrane concentrations cause a greaterportion of wild type VIIa to bind to the membrane. Once again, theincreased function of VIIaQ11E33 was greatest under conditions of lowphospholipid exposure.

Superior coagulation of VIIaQ11E33: Blood clotting assays were conductedat 37° C. using the hand tilt method to detect clot formation. Humanplasma (0.1 mL) was allowed to equilibrate at 37° C. for 1 minute.Various reagents were added in a volume of 0.1 mL of standard buffer.Soluble tissue factor (50 nM) and phospholipid (PS/PC, 10/90, 75 μg/mL)were added to the plasma, along with the factor VIIa concentration shownin FIG. 4 (0.1-32 nM). Finally, 0.1 mL of 25 mM CaCl₂ was added to startthe reaction. Time to form a clot was measured. In most cases, theaverage and standard deviations of replicate samples was reported.

FIG. 4 shows the coagulation times of wild type VIIa versus VIIaQ11E33in normal human plasma. Coagulation was supported by sTF and addedphospholipid vesicles. Endogenous wild type factor VII is approximately10 nM in concentration, and had virtually no impact on coagulationtimes. The background coagulation was 120 seconds, with or without sTF.Factor VIIaQ11E33 showed approximately 8-fold higher activity than thewild type VIIa under these assay conditions. Similar results wereobtained with factor VIII-deficient plasma, suggesting that the majorpathway for blood clotting in this system involved direct activation offactor X by factor VIIa. Overall, factor VIIaQ11E33 was superior to wildtype VIIa in procoagulant activity supported by membrane vesicles andsoluble tissue factor. Wild type zymogen had virtually no activity underthese conditions, as indicated by similar background clotting times of 2minutes, whether or not sTF was added.

Procoagulant activity with normal tissue factor: Activity of VIIa and/orVIIQ11E33 with sTF was measured in normal human plasma. Endogenousfactor VII appeared to have no impact on coagulation time in this assay;the background clotting time was 2 minutes for plasma with or withoutsoluble tissue factor. Soluble tissue factor (50 nM final concentration)and VIIa were added to the plasma before the calcium solution.Coagulation time was assessed for samples containing various levels ofVIIa or VIIaQ11E33. Two human plasma preparations were tested, normaland factor VIII-deficient.

Coagulation supported by normal tissue factor was assayed with standardrabbit brain thromboplastin-HS (HS=high sensitivity) containing calcium(Sigma Chemical Co.). This mixture contains both phospholipids andmembrane-bound tissue factor. Rabbit brain thromboplastin-HS was diluted1:100 in buffer and used in the assay of VII (added in the form ofnormal human plasma, which contains 10 nM factor VII) and VIIQ11E33(added as the pure protein). The thromboplastin (0.2 mL) was added toplasma (0.1 mL) to start the reaction and the time required to form ablood clot was measured. Assays were also conducted with full strengththromboplastin, as described by the manufacturer.

At optimum levels of human thromboplastin, wild type VII showed a normallevel of activity, about 1500 units per mg. This is approximately25-fold less than the activity of wild type factor VIIa (80,000 unitsper mg). The VIIQ11E33 gave approximately 1500-3000 units per mg underthe standard assay conditions, only 2-fold greater than wild type VII.

The difference between wild type VII and VIIQ11E33 was much greater whenthe coagulation conditions were sub-optimal. FIG. 5 shows the clottingtimes and zymogen concentrations in assays that contained 0.01-times thenormal thromboplastin level. Under these conditions, VIIQ11E33 wasapproximately 20-fold more active than wild type factor VII. Thus,greater efficacy of the VIIQ11E33 mutant was especially evident whencoagulation conditions were limited, which is relevant to manysituations in vivo.

Anticoagulant Activities of DEGR-VIIaQ11E33: Standard coagulation assayswere performed with normal human serum and human thromboplastin that wasdiluted 1:10 with buffer. The active site of factor VIIaQ11E33 wasmodified by DEGR as described by Sorenson, B. B. et al., 1997, supra.FIG. 6 shows the clotting time of DEGR-VIIaQ11E33 (0-4 nm) incubatedwith thromboplastin, in calcium buffer, for 15 seconds before additionof the plasma. The time to form a clot was determined with the hand tiltmethod. Clotting time was approximately 45 seconds with about 1 nm ofDEGR-VIIaQ11E33.

Example 2

Purification of Factor VII

Factor VII (wild-type or mutant) was purified by Conconavalin A (Con A),DEAE, and affinity chromatography. Crude media of transfected 293 cellswere incubated with Con A resin (Pharmacia) for four hours at 4° C. Theresin then was washed with a solution containing 50 mM Tris, pH 7.5, 10mM benzamidine, 1 mM CaCl₂, and 1 mM MgCl₂, and factor VII was elutedwith 0.2 M D-methyl mannoside, 0.5 M NaCl in 50 mM Tris buffer, pH 7.5.Factor VII was dialyzed against 50 mM Tris, pH 8.0, 50 mM NaCl, 10 mMbenzamidine, and 25 mM D-methyl mannoside overnight.

Dialyzed factor VII then was incubated with DEAE resin (Pharmacia) forone hour and the mixture was packed into a column. The DEAE column waswashed with 50 mM Tris, pH 8.0, 10 mM Benzamidine, and 50 mM NaCl, andfactor VII was eluted with a gradient from 50 mM to 500 mM NaCl in 50 mMTris buffer, pH 8.0 at a flow rate of 2 mL/min. Fractions containingfactor VII activity were pooled and dialyzed against 50 mM Tris, pH 8.0,50 mM NaCl, and 5 mM CaCl₂ overnight. The Con A and DEAE partiallypurified factor VII was activated by bovine factor Xa (weight ratio1:10, Enzyme Research Laboratory) at 37° C. for one hour.

Activated factor VII was purified further by affinity chromatography. Acalcium-independent monoclonal antibody for factor VII (Sigma) wascoupled to affigel-10 (Bio-Rad Laboratory) as described by Broze et al.,J. Clin. Invest., 1985, 76:937-946, and was incubated with the affinitycolumn overnight at 4° C. The column was washed with 50 mM Tris, pH 7.5,0.1 M NaCl, and factor VIIa was eluted with 50 mM Tris, pH 7.5, 3 MNaSCN at a flow rate of 0.2 mL/min. The eluted fractions wereimmediately diluted five fold into 50 mM Tris, pH 7.5, 0.1 M NaCl.Fractions containing factor VIIa activity were pooled, concentrated, anddialyzed against 50 mM Tris, pH 7.5, 0.1 M NaCl overnight.

The protein concentration of factor VIIa was determined with a Bio-Radprotein assay kit, using BSA as the standard. The purity of factor VIIawas assayed by Coomassie gel and Western Blotting under reduced anddenatured conditions. Proteolytic activity of factor VIIa was measuredusing a synthetic peptide substrate spectrozyme-FVIIa (AmericanDiagnostica) in the presence of thromboplastin (Sigma). Purified factorVIIa was stored in 0.1 mg/ml BSA, 0.1 M NaCl, 50 mM Tris, pH 7.5 at −80°C.

Procoagulant effectiveness of factor VIIa mutants was assessed usingstandard in vitro clotting assays (and modifications thereof);specifically, the prothrombin time (PT) assay and the activated partialthromboplastin (aPTT) assay. Various concentrations of Factor VIIamutants were evaluated in pooled normal human donor plasma and incoagulation factor-deficient (Factor VIII, Factor IX, Factor VII) humanplasmas (Sigma). Clotting times were determined at 37° C. using both aFibroSystem fibrometer (BBL) with a 0.3 mL probe and a Sysmex CA-6000Automated Coagulation Analyzer (Dade Behring).

Platelet poor plasma (PPP) was prepared from pooled normal human donorblood. Blood (4.5 mLs) was drawn from each healthy donor into citrated(0.5 mLs of 3.2% buffered sodium citrate) Vacutainer tubes. Plasma wasobtained after centrifugation at 2,000 g for ten minutes and was kept onice prior to use. Purchased factor-deficient plasmas were reconstitutedaccording to the manufacturer's instructions. Serial dilutions from eachstock of Factor VIIa mutant were all prepared in plasma. All plasmas(with or without Factor VIIa mutants) were kept on ice prior to use. Inall cases, the time to form a clot was measured. The average andstandard deviation of replicate samples was reported.

The prothrombin time (PT) assay was performed in plasmas (with orwithout serial dilutions of added FVIIa mutants) using either of thefollowing PT reagents: Thromboplastin C-Plus (Dade), Innovin (Dade),Thromboplastin With Calcium (Sigma), or Thromboplastin HS With Calcium(Sigma). Assays were conducted according to manufacturer's instructions.In addition to using PT reagents at the manufactured full-strengthconcentration, the PT assay also was performed using various dilutionsof PT reagent. In all cases, the time to form a clot was measured. Theaverage and standard deviation of replicate samples was reported.

The activated partial thromboplastin (aPTT) assay was performed inplasmas (with or without serial dilutions of added Factor VIIa mutants)using either Actin FS (Dade) or APTT reagent (Sigma). Clotting wasinitiated using 0.025M CaCl₂ (Dade) for Actin FS (Dade) or 0.02M CaCl₂(Sigma) for APTT reagent (Sigma). Assays were conducted according tomanufacturer's instructions. In addition to using aPTT reagents at themanufactured full-strength concentration, the aPTT assay also wasperformed using various dilutions of aPTT reagent. In all cases, thetime to form a clot was measured. The average and standard deviation ofreplicate samples was reported.

Clotting assays also were conducted at 37° C. in which differentconcentrations of phospholipid vesicles [at varying ratios of PS/PC orPS/PC/PE (PE=phosphatidylethanolamine)] were added to plasmas (with orwithout serial dilutions of added FVIIa mutants). Clotting was initiatedby the addition of 20 mM CaCl₂. Various reagents were added in standardbuffer. In all cases, the time to form a clot was measured. The averageand standard deviation of replicate samples was reported.

Specific Factor VIIa clotting activity was assessed in plasmas (with orwithout added Factor VIIa mutants) using the STACLOT VIIa-rTF kit(Diagnostica Stago), as per the manufacturer's instructions. This kit isbased on the quantitative clotting assay for activated FVII (Morrisseyet al., 1993, Blood, 81(3):734-744). Clotting times were determinedusing both a FibroSystem fibrometer (BBL) with a 0.3 mL probe and aSysmex CA-6000 Automated Coagulation Analyzer (Dade Behring).

Example 3 Circulatory Time of Factor VIIQ11E33 in the Rat

Two anesthetized (sodium nembutol) Sprague Dawley rats (325-350 g) wereinjected with 36 μg of factor VIIQ11E33 at time zero. Injection wasthrough the juggler vein, into which a cannula had been placed. At thetimes shown in FIG. 7, blood was withdrawn from the carotid artery, intowhich a cannula had been inserted by surgery. The amount of factorVIIQ11E33 in the circulation was estimated from the clotting time ofhuman factor VII-deficient plasma, to which 1 μL of a 1:10 dilution ofthe rat plasma was added. A 1:100 dilution of rabbit brainthromboplastin-HS (Sigma Chemical Co.) was used. Coagulation wasassessed by the manual tube tilt method as described in Example 1. Theamount of factor VII activity in the plasma before injection ofVIIQ11E33 was determined and was subtracted as a blank. Theconcentration of factor VIIQ11E33 in the circulation is given as log nM.A sham experiment in which a third animal received the operation andcannulation but no factor VIIQ11E33 was conducted. The amount of factorVII activity in that animal did not change over the time of theexperiment (100 minutes). At the end of the experiment, the animals wereeuthanized by excess sodium nembutol.

The rats appeared normal throughout the experiment with no evidence ofcoagulation. Therefore, the factor VIIQ11E33 did not causeindiscriminate coagulation, even in the post-operative rat. Thecirculation life-time of the VIIQ11E33 was normal (FIG. 7), withapproximately 40% of the protein being cleared in about 60 minutes andan even slower disappearance of the remaining protein. This was similarto the rate of clearance of bovine prothrombin from the rat. Nelsestuenand Suttie, 1971, Biochem. Biophys. Res. Commun., 45:198-203. This issuperior to wild-type recombinant factor VIIa which gave a circulationhalf-time for functional assays of 20-45 minutes. Thomsen, M. K., etal., 1993, Thromb. Haemost., 70:458-464. This indicated that factorVIIQ11E33 was not recognized as an abnormal protein and that it was notrapidly destroyed by coagulation activity. It appeared as a normalprotein and should have a standard circulation lifetime in the animal.

Example 4 Enhancement of the Membrane Site and Activity of Protein C

Bovine and human protein C show a high degree of homology in the aminoacids of their GLA domains (amino terminal 44 residues), despite about10-fold higher membrane affinity of the human protein. Bovine protein Ccontains a proline at position 11 versus a histidine at position 11 ofhuman protein C. The impact of replacing proline-11 in bovine protein Cwith histidine, and the reverse change in human protein C, was examined.In both cases, the protein containing proline-11 showed lower membraneaffinity, about 10-fold for bovine protein C and 5-fold for humanprotein C. Activated human protein C (hAPC) containing proline atposition 11 showed 2.4 to 3.5-fold lower activity than wild type hAPC,depending on the assay used. Bovine APC containing histidine-11displayed up to 15-fold higher activity than wild type bAPC. Thisdemonstrated the ability to improve both membrane contact and activityby mutation.

Mutagenesis of Protein C: A full-length human protein C cDNA clone wasprovided by Dr. Johan Stenflo (Dept. of Clinical Chemistry, UniversityHospital, Malmö, Sweden). The bovine protein C cDNA clone was providedby Dr. Donald Foster (ZymoGenetics, Inc., USA). The GenBank accessionnumber for the nucleotide sequence of bovine protein C is KO2435, NIDg163486 and is K02059, NID g190322 for the nucleotide sequence of humanprotein C.

Site-directed mutagenesis was performed by a PCR method. For humanprotein C mutagenesis of histidine-11 to proline, the followingoligonucleotides were synthesized: A, 5′-AAA TTA ATA CGA CTC ACT ATA GGGAGA CCC AAG CTT-3′ (SEQ ID NO:8) (corresponding to nucleotides 860-895in the vector pRc/CMV) to create a Hind III site between pRc/CMV andprotein C. B, 5′-GCA CTC CCG CTC CAG GCT GCT GGG ACG GAG CTC CTC CAGGAA-3′ (SEQ ID NO:8) (corresponding to the amino acid residues 4-17 inhuman protein C, the 8th residue in this sequence was mutated from thatfor human protein C to that of bovine protein C, as indicated by theunderline).

For bovine protein C mutagenesis of proline-11 to histidine, thefollowing oligonucleotides were synthesized: A, (as described above); C,5′-ACG CTC CAC GTT GCC GTG CCG CAG CTC CTC TAG GAA-3′ (SEQ ID NO:9)(corresponding to amino acid residues 4-15 in bovine protein C, the 6thamino acid was mutated from that for bovine protein C to that of humanprotein C as marked with underline); D, 5′-TTC CTA GAG GAG CTG CGG CACGGC AAC GTG GAG CGT-3′ (SEQ ID NO:10) (corresponding to amino acidresidues 4-15 in bovine protein C, the 7th amino acid was mutated fromthat for bovine protein C to that of human protein C; mutatednucleotides are underlined); E, 5′-GCA TTT AGG TGA CAC TAT AGA ATA GGGCCC TCT AGA-3′ (SEQ ID NO: 11) (corresponding to nucleotides 984-1019 inthe vector pRc/CMV), creating a Xba I site between pRc/CMV and proteinC).

Both human and bovine protein C cDNAs were cloned into the Hind III andXba I sites of the expression vector pRc/CMV. Human protein C cDNAcontaining the 5′ terminus to amino acid-17 was PCR amplified withintact human protein C cDNA and primers A and B. The volume for the PCRreaction was 100 μl and contained 0.25 μg of template DNA, 200 μM eachof the four deoxyribonucleoside triphosphates, 0.5 mM of each primer and2.5 U of Pwo-DNA polymerase (Boehringer Mannheim) in Tris-HCl buffer (10mM Tris, 25 mM KCl, 5 mM (NH₄)₂SO₄, and 2 mM MgSO₄, pH 8.85). Sampleswere subjected to 30 cycles of PCR consisting of a 2 minute, 94° C.denaturation period, a 2 minute, 55° C. annealing period, and a 2minute, 72° C. elongation period. After amplification, the DNA waselectrophoresed through an 0.8% agarose gel in 40 mM Tris-acetate buffercontaining 1 mM EDTA. PCR products were purified with JET PlasmidMiniprep-Kit (Saveen Biotech AB, Sweden). Human protein C cDNAcontaining respective mutations was cleaved by Hind III and Bsr BI, andthen cloned into pRc/CMV vector that was cleaved by Hind III/Xba I andthat contained human protein C fragment from Bsr BI to the 3′ terminusto produce a human protein C full length cDNA with the mutation.

Bovine protein C cDNA, containing the 5′ terminus through amino acid-11,was PCR amplified with intact human protein C cDNA and primers A and C.Bovine protein C cDNA from amino acid 11 to the 3′ terminus wasamplified with intact human protein C cDNA and primers D and E. Thesetwo cDNA fragments were used as templates to amplify full length bovineprotein C cDNA containing mutated amino acids with primers A and E. PCRreaction conditions were identical to those used for hAPC. The bovineprotein C cDNA containing the respective mutations was cleaved by HindIII and Bsu 36I, and the Hind III/Bsu36I fragment was cloned intopRc/CMV vector containing intact bovine protein C fragments from the Bsu36I to the 3′ terminus to produce full-length bovine protein C cDNAcontaining the mutation. All mutations were confirmed by DNA sequencingprior to transfection.

Cell Culture and Expression: The adenovirus-transfected human kidneycell line 293 was grown in DMEM medium supplemented with 10% fetal calfserum, 2 mM L-glutamine, 100 U/ml of penicillin, 100 U/ml streptomycinand 10 μg/ml vitamin K₁. Transfection was performed using the lipofectinmethod. Felgner, P. L. et al., 1987, Proc. Natl. Acad. Sci. USA,84:7413-7417. Two μg of DNA was diluted to 0.1 mL with DMEM containing 2mM of L-glutamine medium. Ten μL of Lipofectin (1 mg/ml) was added to100 μL of DMEM containing 2 mM L-glutamine medium. DNA and lipofectinwere mixed and left at room temperature for 10-15 min. Cell monolayers(25-50% confluence in 5-cm petri-dishes) were washed twice in DMEM with2 mM L-glutamine medium. The DNA/lipid mixture was diluted to 1.8 mL inDMEM containing 2 mM L-glutamine medium, added to the cells andincubated for 16 hours. The cells were fed with 2 mL of complete mediumcontaining 10% calf serum, left to recover for another 48-72 hours andthen trypsinized and seeded into 10-cm dishes with selection medium(DMEM containing 10% serum and 400 μg/mL of Geneticin) at 1:5. Yan, S.C. B. et al., 1990, Bio/Technology 655-661. Geneticin-resistant colonieswere obtained after 3-5 weeks of selection. Twenty four colonies fromeach DNA transfection were picked, grown to confluence and the mediascreened for protein C expression with a dot-blot assay using monoclonalantibody HPC4 (for human protein C) and monoclonal antibody BPC5 (forbovine protein C). Clones producing high amounts of protein wereisolated and grown until confluence in the presence of 10 μg/mL ofvitamin K₁.

The purification of bovine recombinant protein C and its mutant werebased on the method described previously with some modifications.Rezair, A. R., and Esmon, C. T., 1992, J. Biol. Chem., 267:26104-26109.Conditioned serum-free medium from stably transfected cells wascentrifuged at 5000 rpm at 4° C. for 10 minutes. The supernatant wasfiltered through 0.45 μm of cellulose nitrate membranes (MicroFiltration Systems, Japan). EDTA (5 mM, final concentration) and PPACK(0.2 μM, final concentration) were added to the conditioned medium from293 cells, then passed through a Pharmacia FFQ anion-exchange column atroom temperature using Millipore Con Sep LC100 (Millipore, USA). Theprotein was eluted with a CaCl₂ gradient (starting solution, 20 mMTris-HCl/150 mM NaCl, pH 7.4; limiting solution, 20 mM Tris-HCl/150 mMNaCl/30 mM CaCl₂, pH 7.4). After removal of the CaCl₂ by dialysis andChelex 100 treatment, the protein was reabsorbed to a second FFQ column,then eluted with an NaCl gradient (starting solution 20 mM Tris-HCl/150mM NaCl, pH 7.4; limiting solution, 20 mM Tris-HCl/500 mM NaCl, pH 7.4).At this point in the purification, wild-type and the mutant recombinantbovine protein C were homogeneous as determined by SDS-PAGE.

The first column used for purification of wild-type and mutantrecombinant human protein C was the same as that described for bovineprotein C. The chromatographic method described by Rezair and Esmon wasemployed with some modifications described for the method of protein Spurification. Rezair, A. R., and Esmon, C. T., 1992, supra; He, Z. etal., 1995, Eur. J. Biochem., 227:433-440. Fractions containing protein Cfrom anion-exchange chromatography were identified by dot-blot. Positivefractions were pooled and applied to an affinity column containing theCa²⁺-dependent antibody HPC-4. The column was equilibrated with 20 mMTris-HCl, 150 mM NaCl, pH 7.4, containing 5 mM Benzamidine-HCl and 2 mMCaCl₂. After application, the column was washed with the same buffercontaining 1 M NaCl. Protein C was then eluted with 20 mM Tris-HCl, 150mM NaCl and 5 mM EDTA, pH 7.4, containing 5 mM Benzamidine-HCl. Afterpurification, the purity of all human and bovine recombinant protein Cpreparations was estimated by SDS-PAGE followed by silver staining.Proteins were concentrated using YM 10 filters (Amicon), then dialyzedagainst buffer (50 mM Tris-HCl and 150 mM NaCl, pH 7.4) for 12 hours andstored at −70° C. The concentrations of proteins were measured byabsorbance at 280 nm.

Association of normal and mutant protein C molecules with membranes:LUVs and SUVs were prepared by methods described in Example 1. Lightscattering at 90° to the incident light was used to quantitateprotein-membrane binding as described above for Factor VII (25 μg/mL ofPS/PC, (25/75) at 5 mM calcium (0.05 M Tris buffer-0.1 M NaCl, pH 7.5).

Bovine protein C containing histidine at position 11 interacted withmembranes with about 10-fold higher affinity than wild type protein.When fit to equation 2, the data gave K_(D) values of 930±80 nM forprotein C-H11 and 9200±950 nM for wild type protein C (FIG. 8A). Thedifference in affinity corresponded to about 1.4 kcal/mol at 25° C. Infact, membrane affinity of bovine protein C-H11 was almost identical tothat of native human protein C (660 nM, FIG. 8B). This suggested thatproline 11 formed a major basis for differences between the membranebinding site of human and bovine proteins.

The reverse substitution, replacement of His-11 of human protein C byproline, decreased membrane affinity (FIG. 8B). When fit to equation 2,these data gave K_(D) values of 660±90 nM for wild type human protein Cand 3350±110 nM for human protein C-P11. The impact of prolineintroduction was only slightly less than that of proline in the bovineproteins.

Impact of proline-11 on activity of activated protein C: Activatedprotein C was generated by thrombin cleavage, using identical conditionsfor both the wild type and mutant proteins. Approximately 150 μg of thevarious protein C preparations (1 mg/mL) were mixed with bovine thrombin(3 μg) and incubated at 37° C. for 5 hours. The reaction product wasdiluted to 0.025 M Tris buffer-0.05 M NaCl and applied to a one mLcolumn of SP-Sephadex C-50. The column was washed with one mL of thesame buffer and the flow-through was pooled as activated protein C.Approximately 65-80% of the protein applied to the column was recovered.APC activity was determined by proteolysis of S2366 (0.1 mM) at 25° C.The preparations were compared to standard preparations obtained onlarger scale. Standard human APC was provided by Dr. Walter Kisiel. Forbovine proteins, the standard was a large-scale preparation ofthrombin-activated APC. The activity of bovine APC was consistent forall preparations of normal and mutant proteins (±5%). Two preparationsof bovine APC were used for comparisons. Human APC generated fromthrombin was 55 to 60% as active as the standard. The concentrationsreported in this study were based on activity toward S2366, relative tothat of the standard.

Standard APTT test used bovine or human plasma and standard APTT reagent(Sigma Chemical Co.) according to manufacturers instructions.Alternatively, phospholipid was provided in the form of vesicles formedfrom highly purified phospholipids. In this assay, bovine plasma (0.1mL) was incubated with either kaolin (0.1 mL of 5 mg/mL in 0.05 M Trisbuffer, 0.1 M NaCl, pH 7.5) or ellagic acid (0.1 mM in buffer) for 5minutes at 35° C. Coagulation was started by adding 0.1 mL of buffercontaining phospholipid and the amounts of APC shown, followed by 0.1 mLof 25 mM calcium chloride. All reagents were in standard buffercontaining 0.05 M Tris buffer, 0.1 M NaCl, pH 7.5. An average of a14-fold higher concentration of wild type bAPC was needed to duplicatethe impact of the H11 mutant. Coagulation time at 10 nM bAPC-H11 wasgreater than 120 minutes. Standard APTT reagent (Sigma Chemical Co.)gave a clotting time of about 61 seconds at 35° C. with this plasma.Time required to form a clot was recorded by manual technique. Theamount of phospholipid was designed to be the limiting component in theassay and to give the clotting times shown. The phospholipids used wereSUVs (45 μg/0.4 mL in the final assay, PS/PC, 10/90) or LUVs (120 μg/0.4mL in the final assay, PS/PC, 25/75).

The anticoagulant activity of activated protein C was tested in severalassays. FIG. 9 shows the impact on the APTT assay, conducted withlimiting phospholipid. Under the conditions of this assay, coagulationtimes decreased in a nearly linear, inverse relationship withphospholipid concentration. Approximately 14-times as much wild typebovine APC was needed to equal the effect of bovine APC-H11.

Parts of the study in FIG. 9 were repeated for membranes of PS/PC(25/75, LUV). Again, activity was limited by phospholipid, and itsconcentration was adjusted to give a control clotting time of 360seconds (120 μg of 25% PS in the 0.4 mL assay). Approximately 15-foldmore wild type enzyme was needed to equal the impact of the H11 mutant.Finally, standard APTT reagent (Sigma Chemical Co., standard clottingtime 50±2 seconds) was used. Approximately 10.0±0.7 nM of wild typeenzyme was needed to double the coagulation time to 102±5 seconds. Thesame impact was produced by 2.2±0.1 nM bovine APC-H11. Phospholipid wasnot rate limiting in the standard assay so a smaller impact on membraneaffinity may be expected.

Results for human proteins are shown in FIG. 8B. About 2.5 times as muchhuman APC containing proline-11 was required to prolong coagulation tothe extent of wild type APC. A lower impact of proline-11 introductionmay reflect the smaller differences in membrane affinity of the humanproteins (FIG. 9B).

Inactivation of factor Va: Factor Va inactivation was assayed by themethod of Nicolaes et al., 1996, Thrombosis and Haemostasis, 76:404-410.Briefly, for bovine proteins, bovine plasma was diluted 1000-fold by0.05 M Tris, 0.1 M NaCl, 1 mg/mL bovine serum albumin and 5 mM calciumat pH 7.5. Phospholipid vesicles (5 μg/0.24 mL assay) and 5 μL of 190 nMthrombin were added to activate factor V. After a 10-minute incubationat 37° C., APC was added and the incubation was continued for 6 minutes.Bovine prothrombin (to 10 μM final concentration) and factor Xa (0.3 nMfinal concentration) were added and the reaction was incubated for oneminute at 37° C. A 20 μL sample of this activation reaction was added to0.38 mL of buffer (0.05 M Tris, 0.1 M NaCl, 5 mM EDTA, pH 7.5)containing S2288 substrate (60 μM). The amount of thrombin wasdetermined by the change in absorbance at 405 nM (ε=1.0*10⁴ M⁻¹s⁻¹,k_(cat) for thrombin=100/s). For human proteins, human proteinS-deficient plasma (Biopool Canada, Inc.) was diluted 100-fold, factorVa was activated by human thrombin and the factor Va produced wasassayed with the reagents used for the bovine proteins.

Bovine APC-H11 was 9.2-fold more active than wild type (FIG. 10A) ininactivating factor Va. As for membrane binding (above), the impact ofproline-11 was less with the human proteins, with an average of 2.4-folddifference between the curves drawn for wild type and P-11 mutant (FIG.10B). Similar results were obtained with normal human plasma.

Example 5 Identification of an Archetype Membrane Affinity for theMembrane Contact Site of Vitamin K-Dependent Proteins

Comparison of various human and bovine protein C mutants and othervitamin K-dependent polypeptides led to a proposed membrane contact sitearchetype. The electrostatic archetype consists of an electropositivecore on one surface of the protein, created by bound calcium ions,surrounded by a halo of electronegative charge from amino acids of theprotein. The closer a member of this protein family approaches thiselectrostatic pattern, the higher its affinity for membranes.

Phospholipid vesicles, wild type bovine protein C, protein-membraneinteraction studies, activation and quantitation of protein C, andactivity analysis were as described in Example 4.

Recombinant, mutant protein C was generated by the following procedures.Site-directed mutagenesis was performed by a PCR method. The followingoligonucleotides were synthesized: A, as described in Example 3; F,5′-GCA TTT AGG TGA CAC TAT AGA ATA GGG CCC TCT AGA-3′ (SEQ ID NO:11)(corresponding to nucleotides 984-1019 in the vector pRc/CMV), creatinga Xba I site between pRc/CMV and protein C; G, 5′-GAA GGC CAT TGT GTCTTC CGT GTC TTC GAA AAT CTC CCG AGC-3′ (SEQ ID NO:12) (corresponding toamino acid residues 40-27 in bovine protein C, the 8th and 9th aminoacids were mutated from QN to ED as marked with underline); H, 5′-CAGTGT GTC ATC CAC ATC TTC GAA AAT TTC CTT GGC-3′ (SEQ ID NO:13)(corresponding to amino acid residues 38-27 in human protein C, the 6thand 7th amino acids in this sequence were mutated from QN to ED asindicated with the underline); I, 5′-GCC AAG GAA ATT TTC GAA GAT GTG GATGAC ACA CTG-3′ (SEQ ID NO:14) (corresponding to amino acid residues27-38 in human protein C, the 6th and 7th amino acids in this sequencewere mutated from QN to ED as indicated with underline); J, 5′-CAG TGTGTC ATC CAC ATT TTC GAA AAT TTC CTT GGC-3 (SEQ ID NO:15) (correspondingto amino acid residues 38-27 in human protein C, the 7th amino acids inthis sequence were mutated from Q to E as indicated with underline); K,5′-GCC AAG GAA ATT TTC GAA AAT GTG GAT GAC ACA CTG-3′ (SEQ ID NO:16)(corresponding to amino acid residues 27-38 in human protein C, the 6thamino acid in this sequence was mutated from Q to E as indicated withunderline);

Both bovine and human protein C full length cDNAs were cloned into theHind III and Xba I site of the vector pRc/CMV. To obtain bovine proteinC mutant E33D34, PCR amplification of the target DNA was performed asfollows. Bovine protein C cDNA containing the 5′ terminus to the aminoacid at position 40, was amplified with intact bovine protein C cDNA andprimers A and C. The PCR reaction conditions were as described inExample 4. The sample was subjected to 30 cycles of PCR consisting of a2 min denaturation period at 94° C., a 2 min annealing period at 55° C.and a 2 min elongation period at 72° C. After amplification, the DNA waselectrophoresed through an 0.8% agarose gel in 40 mM Tris-acetate buffercontaining 1 mM EDTA. The PCR products were purified with The GenecleanIII kit (BIO 101, Inc. USA), and the PCR fragment of bovine protein CcDNA containing the respective mutations was cleaved by Hind III and BbsI. The Hind III/Bbs I fragment and the human protein C fragment (BbsI-3′ terminus) were cloned into the Hind II and Xba I sites of pRc/CMVvector to produce a full-length bovine protein C cDNA with themutations. Bovine protein C mutant H11 E33 D34 was created in the sameway, but used bovine protein C mutant H11 as a template in the PCRreaction.

Human protein C cDNA containing the 5′ terminus to amino acid-38 was PCRamplified with intact human protein C cDNA and primers A and D. Humanprotein C cDNA from amino acid 27 to the 3′ terminus was amplified withintact human protein C cDNA and primers B and E. These two cDNAfragments were used as templates to amplify full length bovine protein CcDNA containing mutated amino acids (E33 D34) with primers A and B.Human protein C mutant E33 was obtained by the following steps: humanprotein C cDNA containing the 5′ terminus to amino acid 38 was amplifiedwith intact human protein C cDNA and primers A and F. Human protein CcDNA from amino acid 27 to the 3′ terminus was amplified with intacthuman protein C cDNA and primers B and G. These two cDNA fragments wereused as templates to amplify full length bovine protein C cDNAcontaining mutated amino acids (E33) with primers A and B. The PCRmixture and program were described above. The human protein C PCRproducts containing respective mutations were cleaved by Hind III andSal I, and then the fragment (Hind III-Sal I) together with intact humanprotein C fragment (Sal I-3′ terminus) were cloned into the Hind III andXba I sites of pRc/CMV vector to produce the full length human protein CcDNA with the respective mutations. All mutations were confirmed by DNAsequencing prior to transfection.

The adenovirus-transfected human kidney cell line 293 was cultured andtransfected as described in Example 4. Bovine and human recombinantprotein C and mutants were purified as described in Example 4.

The vitamin K-dependent proteins were classified into four groups on thebasis of their affinities for a standard membrane (Table 4). Sequencesof the amino terminal residues of some relevant proteins including humanprotein C (hC), bovine protein C (bC), bovine prothrombin (bPT), bovinefactor X (bX), and human factor VII (hVII) are given for reference,where X is Gla (γ-carboxyglutamic acid) or Glu.

bPT: ANKGFLXXVRK₁₁GNLXRXCLXX₂₁PCSRXXAFXA₃₁LXSLSATDAF₄₁WAKY (SEQ IDNO:17)

bX: ANS-FLXXVKQ₁₁GNLXRXCLXX₂₁ACSLXXARXV₃₁FXDAXQTDXF₄₁WSKY (SEQ ID NO:18)

hC: ANS-FLXXLRH₁₁SSLXRXCIXX₂₁ICDFXXAKXI₃₁FQNVDDTLAF₄₁WSKH (SEQ ID NO:1)

bC: ANS-FLXXLRP₁₁GNVXRXCSXX₂₁VCXFXXARXI₃₁FQNTXDTMAF₄₁WSFY (SEQ ID NO:2)

hVII: ANA-FLXXLRP₁₁GSLXRXCKXX₂₁QCSFXXARXI₃₁FKDAXRTKLF₄₁WISY (SEQ IDNO:3)

TABLE 4 Charges and Affinity Residue 11 + 29 + 33 + 34 = Sum Total K_(D)(nM) Class I bZ −2 + −2 −1 −4 −6 0.2^(a)-32  hZ −2 + −2 −3 −52.0^(a)-170 Class II bPT-TNBS −2 −2 −1 <10 hVII-Q11E33 + −2 −1 −2 −2  10hS + −2 −1 −2 −2  40 bX + −2 −1 −2 −3  40 bC-E33D34 P + −2 −1 −2 −4 125hX + + −2 −1 −1 −2 160 bPT + −2 −1  0 100 hPT + −2 −1 −1 — bS + + −2  0 0 120 Class III bIX + −2 −1 −1 1000  hIX + −2 −1 −1 1000  hC + +1 −2660 bC-H11 + +1 −1 930 Class IV hC P + +1 −2 3300  hVII P + + −1 +1 +14000  bC P + +1 −1 9200  bVII p + + +1  0 15000  ^(a)Higher affinityvalue equals k_(dissociation)/1*10⁷M⁻¹S⁻¹; the denominator is a typicalk_(association) for other proteins.

In Table 4, vitamin k-dependent polypeptide mutants are in bold. Thetotal charge (residues 1-34) includes 7 calcium ions (+14) and the aminoterminus (+1).

Protein Z was assigned to class I on the basis of its dissociation rateconstant, which was 100 to 1000 times slower than that of otherproteins. If protein Z displayed a normal association rate constant(about 10⁷ M⁻¹s⁻¹) the K_(D) would be about 10⁻¹⁰ M. Wei, G. J. et al.,1982, Biochemistry, 21:1949-1959. The latter affinity may be the maximumpossible for the vitamin K proteins. Class IV proteins differed fromclass III in the presence of proline-11, which may alter affinity bynon-electrostatic means.

While a relatively weak correlation existed between membrane affinityand net negative charge on residues 1-34, an excellent correlation wasfound when only residues 5, 11, 29, 33 and 34 were considered (Table 4).The latter amino acids are located on the surface of the protein. Anumber of proteins were modeled by amino acid substitution into theprothrombin structure and their electrostatic potentials were estimatedby the program DelPhi. A sketch patterned after the electrostaticpotential of bovine protein Z is shown in FIG. 11. Electronegative sitesat 7, 8, 26, 30, 33, 34 and 11 produce a halo of electronegative chargesurrounding a cationic core produced by the calcium-lined pore (FIG.11). The closer a protein structure approaches this structure, thehigher its affinity for the membrane. This correlation is apparent fromthe wild-type proteins, the mutants and chemically modified proteins.

The pattern for other structures can be extrapolated from examination ofthe charge groups that are absent in other proteins. For example, Lys-11and Arg-10 of bovine prothrombin generate high electropositive regionsin their vicinity; the lack of Gla-33 in protein C and Factor VII createless electronegativity in those protein regions. In all cases, highestaffinity corresponded to a structure with an electropositive core thatwas completely surrounded by electronegative protein surface, as shownfor protein Z. The exceptions to this pattern are the proteins withPro-11, which may lower affinity by a structural impact and ser-12(human protein C), which is a unique uncharged residue.

To further test the hypothesis of an archetype for electrostaticdistribution, site-directed mutagenesis was used to replace Gln33Asn34of bovine and human protein C with Glu33Asp34. Glu33 should be furthermodified to Gla during protein processing. These changes altered theelectrostatic potential of bovine protein C to that of bovine factor X.The membrane affinity of the mutant protein was expected to be lowerthan that of factor X due to the presence of proline-11. Indeed, thebovine protein C mutant gave a membrane affinity similar to that ofbovine prothrombin (FIG. 12A), and slightly less than that of bovinefactor X (Table 4).

More interesting was that clot inhibition by APC was greater for themutant than for the wild type enzyme (FIGS. 12B, C). Inclusion ofresults for the P11H mutant of bovine protein C from Example 3 showedthat a family of proteins could be produced, each with differentmembrane affinity and activity, by varying the amino acid substitutionsat positions 11, 33 and 34.

Human protein C mutants containing E33 and E33D34 resulted in a smallincrease in membrane binding affinity (FIG. 13a). Activity of thesemutants was slightly less than the wild-type enzyme (FIG. 13b). Resultswith mutants of bovine protein C suggest that failure of the E33D34mutation in the human protein may arise from H11 and/or other uniqueamino acids in the protein. FIG. 14A shows that the H11 mutant of bovineprotein C bound to the membrane with about 10-fold higher affinity thanwild type protein, the E33D34 mutant bound with about 70-times theaffinity, but that the triple mutant, H11E33D34, was only slightlybetter than the H11 mutant. This relationship was mirrored in theactivity of APC formed from these mutants (FIG. 14B). This resultsuggested that the presence of H11 reduced the impact of E33D34 onmembrane binding affinity.

These results indicated that introduction of E33D34 may not be optimalfor all proteins. Consequently, other mutations may be desirable tocreate human protein C that will use E33D34 and have maximum increasedmembrane affinity. The result with the bovine protein suggested thathistidine 11 may be the primary cause of this phenomenon. Consequently,H11 may be altered to glutamine or to another amino acid in humanprotein C, along with the E33D34 mutation. Another amino acid that mayimpact the affinity is the serine at position 12, an amino acid that isentirely unique to human protein C. These additional changes shouldproduce proteins with enhanced membrane affinity.

The electrostatic archetype was also tested by comparison of human andbovine factor X. The presence of lysine-11 in human factor X suggeststhat it should have lower affinity than bovine factor X. This predictionwas borne out, by the result shown in FIG. 15.

Earlier studies had shown that trinitrobenzenesulfonic acid (TNBS)modification of bovine and human prothrombin fragment 1 had relativelylittle impact (0 to 5-fold) on membrane affinity. Weber, D. J. et al.,1992, J. Biol. Chem., 267:4564-4569; Welsch, D. J. et al., 1988,Biochemistry, 27:4933-4938. Conditions used for the reaction resulted inderivatization of the amino terminus, a change that is linked to loweredmembrane affinity. Welsch, D. J. and Nelsestuen, G. L., 1988,Biochemistry, 27:4939-4945. Protein modification in the presence ofcalcium, which protects the amino terminus, resulted in TNBS-modifiedprotein with much higher affinity for the membrane than native fragment1.

The suggestion that protein Z constitutes the archetype was based on itsdissociation rate constant and that a normal association rate wouldgenerate a K_(D)=10⁻¹ M. Whether this value can be reached is uncertain.It is possible that the slow association rate of protein Z is caused byimproper protein folding, resulting in a low concentration of themembrane-binding conformation. If conditions can be altered to improveprotein folding, association rates of protein Z should improve. Indeed,the association rate constant for protein Z was improved by alterationof pH. The basis for this observation may be related to an unusualfeature of the prothrombin structure which is the close placement of theamino terminus (+1 at pH 7.5) to calcium ions 2 and 3. The +1 charge onthe amino terminus is responsible for the slight electropositive regionjust above Ca-1 in FIG. 11. Charge repulsion between Ca and the aminoterminal may destabilize protein folding and could be a serious problemfor a protein that had low folding stability.

Table 5 provides additional support for the archetype model. It showsthe relationship between distance of ionic groups from strontium ions 1and 8 (corresponding to calcium 1 and an extra divalent metal ion foundin the Sr x-ray crystal structure of prothrombin). The pattern suggeststhat the closer an ionic group is to these metal ions, the higher itsimpact on membrane affinity. The exception is Arg-16, which contributesto the charge of the electropositive core. Higher affinity is correlatedwith electronegative charge at all other sites. This correlation alsoapplies to the GLA residues.

TABLE 5 Distance to Sr-1, 8 and Ion Importance Distance (Å) to:Impact/ion Position Atom^(a) Sr-1 Sr-8 on K_(D)(K_(M)) (A.A-Protein)3(K-PT) ε-N 22.1 21.7 Low or Unknown 5(K-IX) para-C(F) 20.1 20.8 Low orUnknown 19(K/R-VII) C5(L) 20.2 17.8 Low or Unknown 22(K-IX) C4(P) 17.018.5 Low or Unknown 10(R) C6(R) 16.8 12.9 Low or Unknown 25(R-PT) C6(R)11.2 13.8 Low or Unknown 24(X/D-PC) O(S) 8.1 12.0 Low or Unknown11(K-PT,hX,bS; ε-C(K) 14.7 7.4 3-10-fold^(b) Gla-PZ) 33(Gla) γ-C(E) 11.67.5 3—10-fold^(b) 34(D) O(S) 15.3 12.1 3—10-fold^(b) 29(R) para-C(F) 7.58.4 3—10-fold^(b) 16(R) C6(R) 14.2 10.6 3—10-fold^(c) Gla residue^(d)Low importance: 7 12.8 13.3 +2(<2) 15 20 16 <2(<2) 20 19.4 17.8 <2(<2)21 17.2 15 4(3) 33 11.6 7.5 ?^(e)(<2) High importance: 8 8.7 10.9?^(e)(20) 26 3.6 9.5 ?^(e)(50) 17 11.1 9.1 >200(100) 27 8.410.6 >200(85) 30 3.4 4.2 >200(25) ^(a)Distances are from this atom ofbovine prothrombin (residue of prothrombin used in measurement is givenin parentheses) to strontium 1 and 8 of the Sr-Prothrombin fragment 1structure. Seshadri et al. 1994, Biochemistry 33:1087-1092. ^(b)For allbut 16-R, cations lower affinity and anions increase affinity.^(c)Thariath et al. 1997 Biochem. J. 322:309-315. ^(d)Impact of Glu toAsp mutations, distances are averages for the gamma-carboxyl-carbons.K_(D)(K_(M)) data are from Ratcliffe et al. 1993 J. Biol. Chem.268:24339-45. ^(e)Binding was of lower capacity or caused aggregation,making comparisons less certain.

The results in FIG. 15C show that the association rate for protein Z wassubstantially improved at pH 9, where an amino terminal should beuncharged. The rate constant obtained from these data was about 12-foldhigher at pH 9 than at pH 7.5 (FIG. 15C).

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

18 1 44 PRT Homo sapiens MOD_RES (0)...(0) Xaa=gamma carboxyglutamicacid or glutamic acid 1 Ala Asn Ser Phe Leu Xaa Xaa Leu Arg His Ser SerLeu Xaa Arg Xaa 1 5 10 15 Cys Ile Xaa Xaa Ile Cys Asp Phe Xaa Xaa AlaLys Xaa Ile Phe Gln 20 25 30 Asn Val Asp Asp Thr Leu Ala Phe Trp Ser LysHis 35 40 2 44 PRT Bos taurus MOD_RES (0)...(0) Xaa=gammacarboxyglutamic acid or glutamic acid 2 Ala Asn Ser Phe Leu Xaa Xaa LeuArg Pro Gly Asn Val Xaa Arg Xaa 1 5 10 15 Cys Ser Xaa Xaa Val Cys XaaPhe Xaa Xaa Ala Arg Xaa Ile Phe Gln 20 25 30 Asn Thr Xaa Asp Thr Met AlaPhe Trp Ser Phe Tyr 35 40 3 44 PRT Homo sapiens MOD_RES (0)...(0)Xaa=gamma carboxyglutamic acid or glutamic acid 3 Ala Asn Ala Phe LeuXaa Xaa Leu Arg Pro Gly Ser Leu Xaa Arg Xaa 1 5 10 15 Cys Lys Xaa XaaGln Cys Ser Phe Xaa Xaa Ala Arg Xaa Ile Phe Lys 20 25 30 Asp Ala Xaa ArgThr Lys Leu Phe Trp Ile Ser Tyr 35 40 4 44 PRT Bos taurus MOD_RES(0)...(0) Xaa=gamma carboxyglutamic acid or glutamic acid 4 Ala Asn GlyPhe Leu Xaa Xaa Leu Arg Pro Gly Ser Leu Xaa Arg Xaa 1 5 10 15 Cys ArgXaa Xaa Leu Cys Ser Phe Xaa Xaa Ala His Xaa Ile Phe Arg 20 25 30 Asn XaaXaa Arg Thr Arg Gln Phe Trp Val Ser Tyr 35 40 5 45 PRT Homo sapiensMOD_RES (0)...(0) Xaa=gamma carboxyglutamic acid or glutamic acid 5 TyrAsn Ser Gly Lys Leu Xaa Xaa Phe Val Gln Gly Asn Leu Xaa Arg 1 5 10 15Xaa Cys Met Xaa Xaa Lys Cys Ser Phe Xaa Xaa Ala Arg Xaa Val Phe 20 25 30Xaa Asn Thr Xaa Arg Thr Thr Xaa Phe Trp Lys Gln Tyr 35 40 45 6 46 PRTBos taurus MOD_RES (0)...(0) Xaa=gamma carboxyglutamic acid or glutamicacid 6 Tyr Asn Ser Gly Lys Leu Xaa Xaa Phe Val Gln Gly Asn Leu Xaa Arg 15 10 15 Xaa Cys Met Xaa Xaa Lys Cys Ser Phe Xaa Xaa Ala Arg Xaa Val Phe20 25 30 Xaa Asn Thr Xaa Lys Arg Thr Thr Xaa Phe Trp Lys Gln Tyr 35 4045 7 36 DNA Artificial Sequence Protein C mutagenic oligonucleotide 7aaattaatac gactcactat agggagaccc aagctt 36 8 42 DNA Artificial SequenceProtein C mutagenic oligonucleotide 8 gcactcccgc tccaggctgc tgggacggagctcctccagg aa 42 9 36 DNA Artificial Sequence Protein C mutagenicoligonucleotide 9 acgctccacg ttgccgtgcc gcagctcctc taggaa 36 10 36 DNAArtificial Sequence Protein C mutagenic oligonucleotide 10 ttcctagaggagctgcggca cggcaacgtg gagcgt 36 11 36 DNA Artificial Sequence Protein Cmutagenic oligonucleotide 11 gcatttaggt gacactatag aatagggccc tctaga 3612 42 DNA Artificial Sequence Protein C mutagenic oligonucleotide 12gaaggccatt gtgtcttccg tgtcttcgaa aatctcccga gc 42 13 36 DNA ArtificialSequence Protein C mutagenic oligonucleotide 13 cagtgtgtca tccacatcttcgaaaatttc cttggc 36 14 36 DNA Artificial Sequence Protein C mutagenicoligonucleotide 14 gccaaggaaa ttttcgaaga tgtggatgac acactg 36 15 36 DNAArtificial Sequence Protein C mutagenic oligonucleotide 15 cagtgtgtcatccacatttt cgaaaatttc cttggc 36 16 36 DNA Artificial Sequence Protein Cmutagenic oligonucleotide 16 gccaaggaaa ttttcgaaaa tgtggatgac acactg 3617 45 PRT Bos taurus MOD_RES (0)...(0) Xaa=gamma carboxyglutamic acid orglutamic acid 17 Ala Asn Lys Gly Phe Leu Xaa Xaa Val Arg Lys Gly Asn LeuXaa Arg 1 5 10 15 Xaa Cys Leu Xaa Xaa Pro Cys Ser Arg Xaa Xaa Ala PheXaa Ala Leu 20 25 30 Xaa Ser Leu Ser Ala Thr Asp Ala Phe Trp Ala Lys Tyr35 40 45 18 44 PRT Bos taurus MOD_RES (0)...(0) Xaa=gammacarboxyglutamic acid or glutamic acid 18 Ala Asn Ser Phe Leu Xaa Xaa ValLys Gln Gly Asn Leu Xaa Arg Xaa 1 5 10 15 Cys Leu Xaa Xaa Ala Cys SerLeu Xaa Xaa Ala Arg Xaa Val Phe Xaa 20 25 30 Asp Ala Xaa Gln Thr Asp XaaPhe Trp Ser Lys Tyr 35 40

What is claimed is:
 1. A protein C or activated protein C polypeptidecomprising a modified GLA domain, said modified GLA domain comprisingthe amino acid sequence of SEQ ID NO:1 with one, two, three, or fouramino acid substitutions, wherein said substitutions are at positionsselected from residues 10, 11, 28, and
 33. 2. The protein C or activatedprotein C polypeptide of claim 1, wherein said one amino acidsubstitution is at residue
 10. 3. The protein C or activated protein Cpolypeptide of claim 1, wherein said one amino acid substitution is atresidue
 11. 4. The protein C or activated protein C polypeptide of claim1, wherein said one amino acid substitution is at residue
 28. 5. Theprotein C or activated protein C polypeptide of claim 1, wherein saidone amino acid substitution is at residue
 33. 6. The protein C oractivated protein C polypeptide of claim 1, further comprising asubstitution at residue
 32. 7. A protein C or activated protein Cpolypeptide comprising modified GLA domain, said modified GLA domaincomprising the amino acid sequence of SEQ ID NO:1 with three amino acidsubstitutions, wherein said substitutions are at positions selected fromthe group consisting of residues 10, 11, 28, 32, and
 33. 8. The proteinC or activated protein C polypeptide of claim 7, wherein said threeamino acid substitutions are residues 11, 32, and
 33. 9. The protein Cor activated protein C polypeptide of claim 8, wherein residue 32 of SEQID NO:1 is glutamic acid.
 10. The protein C or activated protein Cpolypeptide of claim 8, wherein residue 32 of SEQ ID NO:1 is asparticacid.
 11. The protein C or activated protein C polypeptide of claim 8,wherein residue 32 of SEQ ID NO:1 is glutamic acid and residue 33 of SEQID NO:1 is aspartic acid.
 12. The protein C or activated protein Cpolypeptide of claim 7, wherein residue 11 of SEQ ID NO:1 is glycine,residue 32 of SEQ ID NO:1 is glutamic acid, and residue 33 of SEQ IDNO:1 is aspartic acid.
 13. A protein C or activated protein Cpolypeptide comprising a modified GLA domain, said modified GLA domaincomprising the amino acid sequence of SEQ ID NO:1 with four amino acidsubstitutions, wherein said substitutions are at positions selected fromthe group consisting of residues 10, 11, 28, 32, and
 33. 14. The proteinC or activated protein C polypeptide of claim 13, wherein said fouramino acid substitutions, are at residues 10, 11, 32, and
 33. 15. Theprotein C or activated protein C polypeptide of claim 14, whereinresidue 10 of SEQ ID NO:1 is glutamine residue 11 of SEQ ID NO:1 isglycine, residue 32 of SEQ ID NO:1 is glutamic acid, and residue 33 ofSEQ ID NO:1 is aspartic acid.
 16. A pharmaceutical compositioncomprising said protein C or activated protein C polypeptide of any oneof claims 1-4, 5-11, or 12, 13, 14, 15 and a pharmaceutically acceptablecarrier.
 17. The composition of claim 16 for use in treating thrombosisin a mammal.
 18. The composition of claim 16 for use in decreasing clotformation in a mammal.
 19. The composition of claim 17, wherein saidcomposition is formulated for parenteral administration to a humanpatient.
 20. The composition of claim 18, wherein said composition isformulated for parenteral administration to a human patient.
 21. Anisolated nucleic acid, said nucleic acid comprising a nucleic acidencoding said protein C or activated protein C polypeptide of any one ofclaims 1, 7, or
 13. 22. A method of producing the protein C or activatedprotein C polypeptide of any one of claims 1-4, 5-11, or 15, 13, 14, 16said method comprising expressing an isolated nucleic acid encoding saidprotein C or activated protein C polypeptide in a mammalian host cell.23. The method of claim 22, wherein said mammalian host cell is anadenovirus-transfected human kidney 293 cell.